Methods and compositions for rapid functional analysis of gene variants

ABSTRACT

Methods and compositions are disclosed for rapid functional analysis of gene variants based on analysis of protein-protein and protein-nucleic acid interactions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.61/749,960, filed Jan. 8, 2013, the contents of which are herebyincorporated by reference.

FIELD OF THE INVENTION

The invention relates to methods and compositions for rapid functionalanalysis of gene variants based on protein-protein and protein-nucleicacid interactions.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referred to inparentheses. Full citations for these references may be found at the endof the specification before the claims. The disclosures of thesepublications are hereby incorporated by reference in their entiretiesinto the subject application to more fully describe the art to which thesubject application pertains.

The availability of accurate, relatively low-cost sequencing methods toanalyze the exome or whole genome for novel, rare variants that affectphenotypes has become a game changer in clinical genetics (1).Deciphering the newly identified variants is not a simple task. For anyindividual genome, up to 3.5 million single nucleotide variants and600,000 indels may be identified (2). Similarly for any given exome, upto 17,000 variants may be identified (3). To reduce the complexity ofanalysis, variants are filtered using bioinformatics for rarity bycomparison with catalogs, such as dbSNP and 1000 genomes (3-4). Yet,observing a hit in these catalogs does not negate a possible phenotypiceffect biologically. Current gold standard computational methods, suchas NNSPLICE that predicts splicing alteration (5), and SIFT, SNAP andPolyPhen that predict possible deleterious effects based on conservationof encoded amino acids may also fall short for both sensitivity andspecificity (6-8). Linkage, homozygosity mapping and other purelygenetic methods may lack statistical power from limited number ofaffected individuals within a pedigree or community available for study.Furthermore, demonstration of linkage even at a very high LOD score doesnot preclude the presence of a second variant in linkage disequilibriumthat is in fact causal. The observation that 85% of previouslyidentified causal variants for monogenic disorders were identified inexons or at splice-junction boundaries in introns strongly suggests thatthe vast majority affect the quantity and/or function of the encodedgene RNA and/or protein products (9). In addition, most active proteinsare members of multimeric complexes (10). Thus, mutations in a candidategene may change the quantity of the protein that it encodes, may alterthe post-translational modification of that protein or may affect itsinteraction and localization with its crucial protein binding partnersaltogether. All three of these alterations can be assessed byimmunoassays, which have been a mainstay for quantifying unmodified andmodified proteins for over 30 years (11). These methods include bothimmunohistochemical studies of cells and Western blots of cellhomogenates. Dual immunoassays, such as those provided byco-immunoprecipitation (co-IP) followed by Western blots have becomeimportant for quantifying protein-protein interactions as functionalstudies (12).

The present invention addresses the need for rapid functional analysisof gene variants for phenotype effects based on protein-protein andprotein-nucleic acid interactions and localization.

SUMMARY OF THE INVENTION

A method is provided for multiplex detecting a first protein-secondprotein interaction, in a sample, for up to four distinct firstproteins, first proteins A, B, C and D respectively, the methodcomprising:

contacting the sample with a (i) a first agent attached to a surface ofa magnetic bead that is not labeled with a first primaryoptically-active label, and (ii) a second primary agent attached to asurface of a magnetic bead that is labeled with a first primaryoptically-active label, and (iii) a third primary agent attached to asurface of a non-magnetic bead that is not labeled with a second primaryoptically-active label, and (iv) a fourth primary agent attached to thesurface of a non-magnetic bead that is labeled with a second primaryoptically-active label,wherein the first, second, third and fourth primary agents are differentagents each capable of capturing the distinct first proteins A, B, C andD, respectively;

contacting captured first protein-second protein complex(es) with aplurality of secondary agents each specific for a distinct secondprotein, and each labeled with a separate secondary optically-activelabel wherein the secondary optically-active labels are not the same asthe primary optically-active labels of the primary agents and are eachdistinct from the secondary optically-active label of every other of theoptically-active labeled secondary agents;

recovering magnetic beads complexes from the sample by applying amagnetic field;

recovering non-magnetic bead complexes from the sample based on anon-magnetic physical property of the non-magnetic beads;

passing the recovered magnetic bead complexes through a flow cytometeror optical plate reader;

passing the recovered non-magnetic bead complexes through a flowcytometer or optical plate reader;

detecting the optical signal(s) of the recovered magnetic beadcomplexes; and

detecting the optical signal(s) of the recovered non-magnetic beadcomplexes;

wherein the presence on a magnetic bead complex of only a secondaryoptically-active label indicates the interaction between the firstprotein A and a second protein corresponding to the secondaryoptically-active labeled secondary agent,

and wherein the presence on a magnetic bead complex of both (i) a firstprimary optically-active label and (ii) a secondary optically-activelabel indicates the interaction of the first protein B and a secondprotein corresponding to the secondary optically-active labeledsecondary agent,

and wherein the presence on a non-magnetic bead complex of only asecondary optically-active label indicates the interaction of the firstprotein C and a second protein corresponding to the secondaryoptically-active secondary labeled agent,

and wherein the presence on a non-magnetic bead complex of both (i) asecond primary optically-active label and (ii) a secondaryoptically-active label indicates the interaction of the first protein Dand a second protein corresponding to the secondary optically-activelabeled secondary agent.

Also provided is a method of multiplex detecting a first protein-secondprotein interaction, in a sample, for up to at least four distinct firstproteins, first proteins A, B, C and D respectively, the methodcomprising:

contacting the sample with a (i) a first agent attached to a surface ofa magnetic bead that is not labeled with a first primaryoptically-active label, and (ii) a second primary agent attached to asurface of a magnetic bead that is labeled with a first primaryoptically-active label, and (iii) a third primary agent attached to asurface of a non-magnetic bead that is not labeled with a second primaryoptically-active label, and (iv) a fourth primary agent attached to thesurface of a non-magnetic bead that is labeled with a second primaryoptically-active label,

wherein the first, second, third and fourth primary agents are differentagents each capable of capturing the distinct first proteins A, B, C andD, respectively;

contacting captured first protein-second protein complex(es) with aplurality of secondary agents, each of the plurality being specific fora distinct second protein, and each labeled with a separate secondaryoptically-active label wherein the secondary optically-active labels arenot the same as the primary optically-active labels of the primaryagents and are each distinct from the secondary optically-active labelof every other of the optically-active labeled secondary agents;

recovering magnetic beads complexes from the sample by applying amagnetic field;

recovering non-magnetic bead complexes from the sample based on anon-magnetic physical property of the non-magnetic beads;

passing the recovered magnetic bead complexes through a flow cytometeror optical plate reader:

passing the recovered non-magnetic bead complexes through a flowcytometer or optical plate reader;

detecting the optical signal(s) of the recovered magnetic beadcomplexes; and

detecting the optical signal(s) of the recovered non-magnetic beadcomplexes;

wherein the presence on a magnetic bead complex of only a secondaryoptically-active label indicates the interaction between the firstprotein A and a second protein corresponding to the secondaryoptically-active labeled secondary agent,

and wherein the presence on a magnetic bead complex of both (i) a firstprimary optically-active label and (ii) a secondary optically-activelabel indicates the interaction of the first protein B and a secondprotein corresponding to the secondary optically-active labeledsecondary agent,

and wherein the presence on a non-magnetic bead complex of only asecondary optically-active label indicates the interaction of the firstprotein C and a second protein corresponding to the secondaryoptically-active secondary labeled agent,

and wherein the presence on a non-magnetic bead complex of both (i) asecond primary optically-active label and (ii) a secondaryoptically-active label indicates the interaction of the first protein Dand a second protein corresponding to the secondary optically-activelabeled secondary agent.

Also provided is a method of multiplex detecting a protein-nucleic acidinteraction, in a sample, for up to four distinct proteins, proteins A,B, C and D respectively, the method comprising:

contacting the sample with a (i) a first agent attached to a surface ofa magnetic bead that is not labeled with a first primaryoptically-active label, and (ii) a second primary agent attached to asurface of a magnetic bead that is labeled with a first primaryoptically-active label, and (iii) a third primary agent attached to asurface of a non-magnetic bead that is not labeled with a second primaryoptically-active label, and (iv) a fourth primary agent attached to thesurface of a non-magnetic bead that is labeled with a second primaryoptically-active label,

wherein the first, second, third and fourth primary agents are differentagents each capable of capturing the distinct first proteins A, B, C andD, respectively, under conditions which permit capturing to the primaryagents a first protein-nucleic acid complex from the sample;

contacting captured first protein-nucleic acid complex(es) with aplurality of secondary agents each specific for a distinct nucleic acid,and each labeled with a separate secondary optically-active labelwherein the secondary optically-active labels are not the same as theprimary optically-active labels of the primary agents and are eachdistinct from the secondary optically-active label of every otheroptically-active labeled secondary agent;

recovering magnetic beads complexes from the sample by applying amagnetic field;

recovering non-magnetic bead complexes from the sample based on anon-magnetic physical property of the non-magnetic beads;

passing the recovered magnetic bead complexes through a flow cytometeror optical plate reader;

passing the recovered non-magnetic bead complexes through a flowcytometer or optical plate reader;

quantifying the optical signal(s) of the recovered magnetic beadcomplexes; and

quantifying the optical signal(s) of the recovered non-magnetic beadcomplexes;

wherein the presence on a magnetic bead complex of only a secondaryoptically-active label indicates the interaction between the firstprotein A and a nucleic acid corresponding to the secondaryoptically-active labeled secondary agent,

and wherein the presence on a magnetic bead complex of both (i) a firstprimary optically-active label and (ii) a secondary optically-activelabel indicates the interaction of the first protein B and a nucleicacid corresponding to the secondary optically-active labeled secondaryagent,

and wherein the presence on a non-magnetic bead complex of only asecondary optically-active label indicates the interaction of the firstprotein C and a nucleic acid corresponding to the secondaryoptically-active secondary labeled agent,

and wherein the presence on a non-magnetic bead complex of both (i) asecond primary optically-active label and (ii) a secondaryoptically-active label indicates the interaction of the first protein Dand a nucleic acid corresponding to the secondary optically-activelabeled secondary agent.

The invention provides methods of analyzing a gene variant based onendogeneous or transient protein-protein interaction, the methodcomprising: attaching a first primary agent to the surface of magneticbeads that are not labeled with an optically active label, such as forexample, but not limited to, a fluorescent label, and attaching a secondprimary agent to the surface of magnetic beads that are labeled with anoptically active label; attaching a third primary agent to the surfaceof non-magnetic beads that are not labeled with an optically activelabel and attaching a fourth primary agent to the surface ofnon-magnetic beads that are labeled with an optically active label,wherein the first, second, third and fourth primary agents are differentagents and wherein the first, second, third and fourth primary agentsare each capable of capturing a distinct protein complex from a cell ortissue lysate; capturing to the primary agents a protein complex from acell or tissue lysate, where the protein complex comprises a protein ofinterest that is a product of a gene or a gene variant and where theprotein of interest is part of a complex with another protein; probingthe protein-protein complex with one or more optically active labeledsecondary agents specific for a member of the complex; wherein the sameone or more optically active labels can be used to label secondaryagents on any of i) the magnetic beads that are not labeled with anoptically active label, ii) the magnetic beads that are labeled with anoptically active label, iii) the non-magnetic beads that are not labeledwith an optically active label, and iv) the non-magnetic beads that arelabeled with an optically active label; separating magnetic beads fromthe lysate based on magnetic properties of the magnetic beads;separating non-magnetic beads from the lysate based on a physicalproperty of the non-magnetic beads; and measuring optical activity ofoptically active agents, wherein the absence or presence of theoptically active label on the magnetic beads is used as an identifier todistinguish optically active protein complexes captured by the first andsecond primary agents, respectively, and wherein the absence or presenceof the optically active label on the non-magnetic beads is used as anidentifier to distinguish optically active protein complexes captured bythe third and fourth primary agents, respectively.

The invention also provides methods of analyzing a gene variant based onendogenous or transient protein-nucleic acid interaction, the methodcomprising: attaching a first primary agent to the surface of magneticbeads that are not labeled with an optically active label and attachinga second primary agent to the surface of magnetic beads that are labeledwith an optically active label; attaching a third primary agent to thesurface of non-magnetic beads that are not labeled with an opticallyactive label and attaching a fourth primary agent to the surface ofnon-magnetic beads that are labeled with an optically active label,wherein the first, second, third and fourth primary agents are differentagents and wherein the first, second, third and fourth primary agentsare each capable of capturing a distinct protein-nucleic acid complexfrom a cell or tissue lysate; capturing to the primary agents aprotein-nucleic acid complex from a cell or tissue lysate, where theprotein-nucleic acid complex comprises a gene or a gene variant nucleicacid sequence; separating magnetic beads from the lysate based onmagnetic properties of the magnetic beads; separating non-magnetic beadsfrom the lysate based on a physical property of the non-magnetic beads;optionally digesting nucleic acids with nucleases prior to digestingproteins on protein-nucleic acid complexes to release nucleic acids; andamplifying the released nucleic acids; wherein the absence or presenceof the optically active label on the magnetic beads is used todistinguish optically active protein-nucleic complexes captured by thefirst and second primary agents, respectively, and wherein the absenceor presence of the optically active label on the non-magnetic beads isused to distinguish optically active protein-nucleic acid complexescaptured by the third and fourth primary agents, respectively.

The invention effectively incorporate methods for optimal detection andselection of various targets (more than two) with limited startingbiomaterial. The invention includes a gating principle thatsubstantially increases sensitivity of the detection of flowcytometry-based immunoassay with no amplification steps of any sort.

The invention provides kits for identification of the protein-proteininteractions, protein-nucleic acid interactions, cell-based proteinexpression, protein modifications, localization and a standardconcurrent immunoprecipitation (IP)-Western. The kits allow a simplifiedflow-based immunoassay that is truly high-throughput and unified sampleprocessing techniques. The kits contain optimized comprehensivechemistries and significant improvement over traditional methods ofIP-Western blots.

The kit provides versatility to perform multiple assays, assays such asDigital Cell Western (DCW), rapid assessment of protein-proteininteraction and localization by modified flow cytometry-based IP,simplified protein-nucleic acid interactions assessment and capturednucleic acid purification for massive parallel sequencing (MPS),genotyping and polymerase chain reactions (PCR) applications.

DCW, a form of digital Western in this kit that is designed andoptimized to probe individual cells for various target proteinexpressions including post-translational modifications that can beeffortlessly detected simultaneously allowing thousands of data pointsfrom each fixed cell samples to be aggregated as digital calculation forstatistical power.

The invention utilizes a combination of bar-coded bead system, forexample but not limited to surface enhanced Dynabeads and Carboxylmodified beads (CML) to detect multiple variant protein interactionsfrom a single lysate. With the paramagnetic properties of Dynabeads,first separation phase allows clearance of targets bound to theDynabeads by magnetic separation, remaining supernatant containing CMLbeads will capture another set of targets that will be separated bycentrifugal force. This approach using bar-coded bead systems allowsdoubling of the number of detected targets with this methodology invarious high-throughput formats.

The invention allows assessment of protein-nucleic acid interactions andpurification of bound nucleic acids sequences, which is suitable for usein Chromatin Immunoprecipitation (CHIP), RNA IP PCR, genotyping andsequencing (MPS). This is first of its kind kit that allows assessmentof variant protein interactions and simultaneous purification of anybinding nucleic acid sequences to the variant proteins. Applicationssuch as CHIP, CHIP-sequencing, and RNA IP are transformed into astreamlined assay suitable for large-scaled investigation of varioustargets in research and diagnostic applications.

Also provided is a method of multiplex detecting a protein-nucleic acidinteraction, in a sample, for up to at least four distinct proteins,proteins A, B, C and D respectively, the method comprising:

a) contacting the sample with a (i) a first agent attached to a surfaceof a magnetic bead that is not labeled with a first primaryoptically-active label, and (ii) a second primary agent attached to asurface of a magnetic bead that is labeled with a first primaryoptically-active label, and (iii) a third primary agent attached to asurface of a non-magnetic bead that is not labeled with a second primaryoptically-active label, and (iv) a fourth primary agent attached to thesurface of a non-magnetic bead that is labeled with a second primaryoptically-active label,

wherein the first, second, third and fourth primary agents are differentagents each capable of capturing the distinct first proteins A, B, C andD, respectively, under conditions which permit capturing to the primaryagents a first protein-nucleic acid complex from the sample;

b) optionally, contacting one or more of the four distinct proteins ofthe sample with one or more nucleic acids either prior to a) orsubsequent to a);

c) recovering magnetic beads complexes from the sample by applying amagnetic field and recovering non-magnetic bead complexes from thesample based on a non-magnetic physical property of the non-magneticbeads;

d) contacting one or more of (i) the magnetic beads complexes not havinga first primary optically-active label; (ii) the magnetic beadscomplexes having a first primary optically-active label; (iii) thenon-magnetic beads complexes not having a first primary optically-activelabel; (iv) the non-magnetic beads complexes having a first primaryoptically-active label, with optional nuclease then a Proteinase K so asto digest the proteins thereon and release any nucleic acids boundthereto;

e) sequencing nucleic acid(s) released in step d)(i) so as to therebyidentify the nucleic acids that have interacted with the first distinctprotein; in step dxii) so as to thereby identify the nucleic acids thathave interacted with the second distinct protein; in step d)(iii) so asto thereby identify the nucleic acids that have interacted with thethird distinct protein; and/or in step dxiv) so as to thereby identifythe nucleic acids that have interacted with the fourth distinct protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Application areas of Functional Variant Assay (FVA) kit. The kitprovides versatility using flow cytometry to perform multiple assays,such as Digital Cell Western (DCW) concurrent with traditional IPwestern, rapid assessment of protein-protein interactions of varioustarget using limited biomaterial, protein-nucleic acid interactionsassessment, bound nucleic acid purification and enrichment for MPS,genotyping and polymerase chain reactions (PCR) applications. DCW is aform of digital western in this kit that is designed and optimized toprobe individual cells for various target protein expressions includingpost-translational modifications can be effortlessly detected, whilesimultaneously measuring thousands of data points from each of the fixedcell samples; data points then can be aggregated as digital calculationsfor statistical power.

FIG. 2. Examples of application of the invention. The invention allowsassessment by modified flow immunoprecipitation of protein-proteininteractions, protein-nucleic acid interactions and purification withenrichment of bound/captured nucleic acids sequences suitable for use inPCR, genotyping and sequencing (MPS). The kit allows concurrentassessment of variant protein interactions, digital cell western andsimultaneous purification of any binding nucleic acid sequences to thevariant proteins for MPS. Applications such as ChromatinImmunoprecipitation (CHIP), CHIP-sequencing, and RNA IP are transformedinto a streamlined assay suitable for large-scaled investigation ofvarious targets in research and diagnostic applications.

FIG. 3. Example of scheme of the present invention. The inventionutilizes a combination of beads, for example but not limited to epoxyDynabeads and Carboxyl modified beads (CML) to detect multiple variantproteins. With the paramagnetic properties of Dynabeads, a firstseparation phase allows clearance of targets bound to the Dynabeads bymagnetic separation, remaining supernatant containing CML beads willcapture another set of targets that will be separated by centrifugalforce. This approach using two-bead system will double the number ofdetected targets using existing methodology optimized forhigh-throughput analysis. Bar coding complexes to identify differentprotein-protein complexes or different protein-nucleic acid complexes onbeads with different optically active labels (“vertical bar coding”).Another form of bar-coding means to identify different analytes on themagnetic beads and/or non-magnetic beads using optically active labels,for example but not limited to fluorescent labels (“horizontal barcoding”). For example but not limited to, for 7 different fluorescentlabels, one label can be used to identify a portion and/or subset ofmagnetic or non-magnetic beads and the remaining 6 labels can be used tolabel different protein targets or different protein-nucleic acidcomplexes attached to the beads. This method is expandable depending onthe capabilities and the number of available lasers on the flowcytometer or plate readers.

FIG. 4A-4C. Functional Variant Assay (FVA) performed on B-lymphoblastoidcells from wild-type and p.Leu189Arg using the MAP3K1 bait antibody andthe Alexa 647 and Alexa 488-labeled MAP3K4 target antibodies. A. Theflow cytometry gated results shows increased binding of MAP3K4 to mutantMAP3K1 as shown previously by standard methods. B. Results compiled fromthree independent experiments for each pathogenic mutation, Leu189Arg,p.Leu189Pro and c.634-8A show increased MAP3K4 binding to mutant MAP3K1(p<0.05). C. Conventional IP Western blots of primary B-lymphoblastoidcells detected an approximate 2-fold increase of binding of MAP3K4 toMAP3K1 from all three mutant cell lines compared to wild-type. Loadingcontrol is actin and input control MAP3K1 on the lowest panel.

FIG. 5A-5C. Reverse FVA performed on B-lymphoblastoid cells fromwild-type and p.Leu189Arg using the RHOA bait antibody and the Alexa488-labeled MAP3K1 target antibody. A. The flow cytometry gated resultsshows increased binding of mutant MAP3K1 to RHOA complexes. B. Resultscompiled from three independent experiments for each pathogenicmutation, Leu189Arg, p.Leu189Pro and c.634-8A, show increased mutantMAP3K1 binding to RHOA (p<0.05). C. Conventional IP Western blots ofprimary B-lymphoblastoid cells detect an approximate 2.5-fold increaseof binding of mutant MAP3K1 to RHOA from all three mutant cell linescompared to wild-type.

FIG. 6A-6C. A. Individual sample intensities of MAP3K1 input prior topull-down for MAP3K4 for FIG. 4C were quantified from conventionalWestern blots using the Licor Software 3.0. B. After controlling forMAP3K1 loading the intensities were further normalized to actin, showingan average 2-fold increase of MAP3K4 binding in all mutant samples. C.Reverse IP using RHOA as bait shows increased binding of MAP3K1 to allmutant samples, about 2.5-fold increase compared to WT samples. Theseresults were normalized to histone as a loading control.

FIG. 7A-7B. A. After treatment with Etoposide and UV radiation or B. theX-Ray mimetic drug, Bleomycin, the localization of BRCA 1 to nuclearfoci was markedly lower among mutant samples compared to normal samplesas measured with the present assay.

FIG. 8. Traditional phospho-Western vs. Digital Cell phospho-Western.Preparation and run: 2 days vs. less than 2 hours. TraditionalIP-western vs. Flow Variant Analysis: Preparation and run time: 5 daysvs. 5 hours.

DETAILED DESCRIPTION OF THE INVENTION

A method is provided of multiplex detecting a first protein-secondprotein interaction, in a sample, for up to at least four distinct firstproteins, first proteins A, B, C and D respectively, the methodcomprising:

contacting the sample with a (i) a first agent attached to a surface ofa magnetic bead that is not labeled with a first primaryoptically-active label, and (ii) a second primary agent attached to asurface of a magnetic bead that is labeled with a flint primaryoptically-active label, and (iii) a third primary agent attached to asurface of a non-magnetic bead that is not labeled with a second primaryoptically-active label, and (iv) a fourth primary agent attached to thesurface of a non-magnetic bead that is labeled with a second primaryoptically-active label,

wherein the first, second, third and fourth primary agents are differentagents each capable of capturing the distinct first proteins A, B, C andD, respectively;

contacting captured first protein-second protein complex(es) with aplurality of secondary agents each specific for a distinct secondprotein, and each labeled with a separate secondary optically-activelabel wherein the secondary optically-active labels are not the same asthe primary optically-active labels of the primary agents and are eachdistinct from the secondary optically-active label of every other of theoptically-active labeled secondary agents;

recovering magnetic beads complexes from the sample by applying amagnetic field;

recovering non-magnetic bead complexes from the sample based on anon-magnetic physical property of the non-magnetic beads;

passing the recovered magnetic bead complexes through a flow cytometeror optical plate reader and quantifying the optical signal emittedtherefrom;

passing the recovered non-magnetic bead complexes through a flowcytometer or optical plate reader and quantifying the optical signalemitted therefrom;

detecting the optical signal(s) of the recovered magnetic beadcomplexes; and

detecting the optical signal(s) of the recovered non-magnetic beadcomplexes;

wherein the presence on a magnetic bead complex of only a secondaryoptically-active label indicates the interaction between the firstprotein A and a second protein corresponding to the secondaryoptically-active labeled secondary agent,

and wherein the presence on a magnetic bead complex of both (i) a firstprimary optically-active label and (ii) a secondary optically-activelabel indicates the interaction of the first protein B and a secondprotein corresponding to the secondary optically-active labeledsecondary agent,

and wherein the presence on a non-magnetic bead complex of only asecondary optically-active label indicates the interaction of the firstprotein C and a second protein corresponding to the secondaryoptically-active secondary labeled agent, and wherein the presence on anon-magnetic bead complex of both (i) a second primary optically-activelabel and (ii) a secondary optically-active label indicates theinteraction of the first protein D and a second protein corresponding tothe secondary optically-active labeled secondary agent.

A method of multiplex detecting a first protein-second proteininteraction, in a sample, for up to at least four distinct firstproteins, first proteins A, B, C and D respectively, the methodcomprising:

contacting the sample with a (i) a first agent attached to a surface ofa magnetic bead that is not labeled with a first primaryoptically-active label, and (ii) a second primary agent attached to asurface of a magnetic bead that is labeled with a first primaryoptically-active label, and (iii) a third primary agent attached to asurface of a non-magnetic bead that is not labeled with a second primaryoptically-active label, and (iv) a fourth primary agent attached to thesurface of a non-magnetic bead that is labeled with a second primaryoptically-active label,

wherein the first, second, third and fourth primary agents are differentagents each capable of capturing the distinct first proteins A, B, C andD, respectively;

contacting captured first protein-second protein complex(es) with aplurality of secondary agents, each of the plurality being specific fora distinct second protein, and each labeled with a separate secondaryoptically-active label wherein the secondary optically-active labels arenot the same as the primary optically-active labels of the primaryagents and are each distinct from the secondary optically-active labelof every other of the optically-active labeled secondary agents;

recovering magnetic beads complexes from the sample by applying amagnetic field;

recovering non-magnetic bead complexes from the sample based on anon-magnetic physical property of the non-magnetic beads;

passing the recovered magnetic bead complexes through a flow cytometeror optical plate reader;

passing the recovered non-magnetic bead complexes through a flowcytometer or optical plate reader;

detecting the optical signal(s) of the recovered magnetic beadcomplexes; and

detecting the optical signal(s) of the recovered non-magnetic beadcomplexes;

wherein the presence on a magnetic bead complex of only a secondaryoptically-active label indicates the interaction between the firstprotein A and a second protein corresponding to the secondaryoptically-active labeled secondary agent,

and wherein the presence on a magnetic bead complex of both (i) a firstprimary optically-active label and (ii) a secondary optically-activelabel indicates the interaction of the first protein B and a secondprotein corresponding to the secondary optically-active labeledsecondary agent,

and wherein the presence on a non-magnetic bead complex of only asecondary optically-active label indicates the interaction of the firstprotein C and a second protein corresponding to the secondaryoptically-active secondary labeled agent,

and wherein the presence on a non-magnetic bead complex of both (i) asecond primary optically-active label and (ii) a secondaryoptically-active label indicates the interaction of the first protein Dand a second protein corresponding to the secondary optically-activelabeled secondary agent.

Such methods may be termed “PrCo-IP.” In an embodiment, the plurality ofsecondary agents is up to twelve secondary agents. In an embodiment, theplurality of secondary agents is up to twenty-four secondary agents. Inan embodiment, the plurality of secondary agents is up to thirty-sixsecondary agents. In an embodiment, the plurality of secondary agentsis, not limited to, up to thirty-eight secondary agents. The method maybe performed with as many types of secondary agents as are discretelydistinguishable.

The method can further comprise multiplex detecting more than fourdistinct proteins. For detecting n distinct proteins, the magnetic andnon-magnetic bead populations must comprise between them a primary agentfor each of the n proteins and at least n−2 primary optically-activelabels, one for each of n−2 of the proteins. The remaining 2 proteinscan be detected by the magnetic and non-magnetic beads which having theprimary agent for each of those proteins, but which are unlabeled withthe primary optically-active agents.

A method of multiplex detecting protein-nucleic acid interactions in asample for up to four distinct proteins, proteins A, B, C and Drespectively, the method comprising: a) contacting the sample with a (i)a first agent attached to a surface of a magnetic bead that is notlabeled with a first primary optically-active label, and (ii) a secondprimary agent attached to a surface of a magnetic bead that is labeledwith a first primary optically-active label, and (iii) a third primaryagent attached to a surface of a non-magnetic bead that is not labeledwith a second primary optically-active label, and (iv) a fourth primaryagent attached to the surface of a non-magnetic bead that is labeledwith a second primary optically-active label,

wherein the first, second, third and fourth primary agents are differentagents each capable of capturing the distinct proteins A, B, C and D,respectively, under conditions which permit capturing to the primaryagents a first protein-nucleic acid complex from the sample:

b) recovering magnetic beads complexes from the sample by applying amagnetic field and recovering non-magnetic bead complexes from thesample based on a non-magnetic physical property of the non-magneticbeads:

c) contacting one or more of (i) the magnetic bead complexes not havinga first primary optically-active label; (ii) the magnetic bead complexeshaving a first primary optically-active label; (iii) the non-magneticbead complexes not having a first primary optically-active label; (iv)the non-magnetic bead complexes having a first primary optically-activelabel, with a Proteinase K so as to digest the proteins thereon andrelease any nucleic acids bound thereto;

d) sequencing nucleic acid(s) released in step c)(i) so as to therebyidentify the nucleic acids that have interacted with distinct protein A;in step c)(ii) so as to thereby identify the nucleic acids that haveinteracted with distinct protein B; in step c)(iii) so as to therebyidentify the nucleic acids that have interacted with distinct protein C;and/or in step cxiv) so as to thereby identify the nucleic acids thathave interacted with distinct protein D.

Such a method may be termed “NACo-IP.” In an embodiment, the methodfurther comprises contacting one or more of the four distinct proteinsof the sample with one or more nucleic acids either prior to a) orsubsequent to a). It is apparent that in some circumstances the samplewill contain proteins-nucleic acid interactions prior to application ofthe method in which case such a step is not required. In other cases,where the protein(s) of the sample are free of nucleic interaction priorto application of the method, nucleic acids can be contacted with theproteins to determine if the nucleic acids interact with the proteins.In an embodiment, the plurality of secondary agents is up to twelvesecondary agents. In an embodiment, the plurality of secondary agents isup to twenty-four secondary agents. In an embodiment, the plurality ofsecondary agents is up to thirty-six secondary agents. In an embodiment,the plurality of secondary agents is up to thirty-eight secondaryagents. The method may be performed with as many types of secondaryagents as are discretely distinguishable.

The method can further comprise multiplex detecting more than fourdistinct protein-nucleic acid interactions. For detecting n distinctprotein-nucleic acid interactions, the magnetic and non-magnetic beadpopulations must comprise between them a primary agent for each of the nproteins.

In an embodiment, the method further comprises digesting the proteins ofthe protein-nucleic acids with a Proteinase K. In an embodiment, themethod further comprises amplifying the nucleic acids released afterProteinase K digestion. In an embodiment, the method is used to identifygenetic heterogeneity among a population of cells. In an embodiment, thesample contacted with the primary agents is subsequently contacted witha nuclease to digest nucleic acids that are not interacting/bound to aprotein of the sample. In an embodiment, the method further comprisesprobing the protein-nucleic acid complex(es) with one or more opticallyactive secondary agents each specific for one of distinct first proteinsA, B, C and D, so as to identify bead complexes comprising a bead, adistinct protein and a primary agent, and recovering such beadcomplexes. In an embodiment, the method further comprises comprisingafter step c) and before step d) passing the recovered magnetic beadcomplexes through a flow cytometer or optical plate reader and passingthe recovered non-magnetic bead complexes through a flow cytometer oroptical plate reader; and detecting the optical signal(s) of therecovered magnetic bead complexes and detecting the optical signal(s) ofthe recovered non-magnetic bead complexes and, optionally, quantifyingthe optical signal(s) detected so as to thereby quantify the amount ofprotein-nucleic acid interaction on the bead.

In an embodiment, the method further comprises amplifying the nucleicacids released after contacting with a Proteinase K, but prior tosequencing.

Also provided is a method of multiplex detecting a protein-nucleic acidinteraction, in a sample, for up to four distinct proteins, proteins A,B, C and D respectively, the method comprising:

contacting the sample with a (i) a first agent attached to a surface ofa magnetic bead that is not labeled with a first primaryoptically-active label, and (ii) a second primary agent attached to asurface of a magnetic bead that is labeled with a first primaryoptically-active label, and (iii) a third primary agent attached to asurface of a non-magnetic bead that is not labeled with a second primaryoptically-active label, and (iv) a fourth primary agent attached to thesurface of a non-magnetic bead that is labeled with a second primaryoptically-active label,

wherein the first, second, third and fourth primary agents are differentagents each capable of capturing the distinct first proteins A, B, C andD, respectively, under conditions which permit capturing to the primaryagents a first protein-nucleic acid complex from the sample;

contacting captured first protein-nucleic acid complex(es) with aplurality of secondary agents each specific for a distinct nucleic acid,and each labeled with a separate secondary optically-active labelwherein the secondary optically-active labels are not the same as theprimary optically-active labels of the primary agents and are eachdistinct from the secondary optically-active label of every otheroptically-active labeled secondary agent;

recovering magnetic beads complexes from the sample by applying amagnetic field;

recovering non-magnetic bead complexes from the sample based on anon-magnetic physical property of the non-magnetic beads;

passing the recovered magnetic bead complexes through a flow cytometeror optical plate reader and quantifying the optical signal emittedtherefrom;

passing the recovered non-magnetic bead complexes through a flowcytometer or optical plate reader and quantifying the optical signalemitted therefrom;

quantifying the optical signal(s) of the recovered magnetic beadcomplexes; and

quantifying the optical signal(s) of the recovered non-magnetic beadcomplexes;

wherein the presence on a magnetic bead complex of only a secondaryoptically-active label indicates the interaction between the firstprotein A and a nucleic acid corresponding to the secondaryoptically-active labeled secondary agent,and wherein the presence on a magnetic bead complex of both (i) a firstprimary optically-active label and (ii) a secondary optically-activelabel indicates the interaction of the first protein B and a nucleicacid corresponding to the secondary optically-active labeled secondaryagent,and wherein the presence on a non-magnetic bead complex of only asecondary optically-active label indicates the interaction of the firstprotein C and a nucleic acid corresponding to the secondaryoptically-active secondary labeled agent,and wherein the presence on a non-magnetic bead complex of both (i) asecond primary optically-active label and (ii) a secondaryoptically-active label indicates the interaction of the first protein Dand a nucleic acid corresponding to the secondary optically-activelabeled secondary agent.

In an embodiment of the methods, the presence on a bead complex of afirst primary optically-active label and/or a secondary optically-activelabel is determined by quantifying the optical signal thereof.

In an embodiment of the methods, the optical signal is collected withone or more photomultipliers. In an embodiment of the methods therecovered bead complexes are passed through a flow cytometer. In anembodiment of the methods the recovered bead complexes are passedthrough an optical plate reader.

In an embodiment of the methods, the methods further comprisequantifying the optical signal(s) detected and comparing the quantifiedamount against a control amount or control curve so as to therebyquantify the amount of first protein-second protein interaction on thebead or the amount of protein-nucleic acid interaction on the bead, asrelevant. Such a method may be termed “digital cell Western” or “DCW.”

In an embodiment of the methods, each primary agent comprises anantibody or comprises an antigen-binding fragment of an antibody.

In an embodiment of the methods, each secondary agent comprises anantibody or comprises an antigen-binding fragment of an antibody. In anembodiment of the methods, the antibodies are monoclonal antibodies. Inan embodiment of the methods, the antibody fragments are F(ab′)2fragments, Fab′ fragments or ScFvs.

In an embodiment of the methods, the primary agents comprises anantibody or comprise an antigen-binding fragment of an antibody, andwherein the secondary agents are oligonucleotides that hybridize with aspecific sequence of a nucleic acid.

In an embodiment of the methods, the sample is a cell or tissue lysate,or any biological lysates. In an embodiment of the methods, the sampleis a cell, and the cell is fixed and permeabilized so as to permitprimary and second agent entry into the cell prior to performing themethod.

In an embodiment of the methods, the first protein-second proteincomplex comprises a protein of that is a product of a gene variant.

In an embodiment of the methods, the first proteins A, B, C and D aredistinct variant forms of a single protein.

In an embodiment of the methods, the sample contains at least one firstdistinct protein-second distinct protein interaction.

In an embodiment of the methods, a bead complex comprises the beadhaving a primary agent attached thereto, wherein the primary agent isattached to a distinct first protein and the distinct first protein isinteracting (for example, bound to) a distinct second protein which hasbound a labeled secondary agent. In an embodiment of the methods, afirst protein-second protein complex is an association of the firstprotein and second protein (for example, by way of binding to eachother).

In an embodiment, the interaction is an intermolecular interaction,occurring by intermolecular forces, such as ionic bonds, hydrogen bondsor van der Waals forces.

A “distinct” protein is one that has a sequence which is non-identicalto every other recited “distinct” protein. The distinct proteinsreferred to herein are distinct in that they have different amino acidsequences. The distinct proteins can be variants, or can be completelydifferent proteins. “Proteins A, B, C and D,” or grammatical variationsthereof, as referred to herein are not actual protein names, but merelyidentifiers to distinguish up to four different proteins.

In an embodiment the term “variant,” for example, of a gene or aprotein, means one having 97%, 98% or 99% or greater (but not 100%)sequence identify with the gene or protein, respectively, that therecited gene or protein is a variant of. In an embodiment the term“variant,” for example, of a gene or a protein, means one having 99% orgreater (but not 100%) sequence identify with the gene or protein,respectively, that the recited gene or protein is a variant of.

In an embodiment of the methods, forward scatter (FSC) and/or sidescatter (SSC) are adjusted with a control un-complexed bead populationprior to initiating the method so as to permit complexed beads to bedetected.

In an embodiment of the methods, the ratio of primary agent to primaryoptically-active labels on primary agents so-labeled is 1:1. In anembodiment of the methods, the ratio of secondary agent to secondaryoptically-active labels on secondary agents so-labeled is 1:1.

In an embodiment of the methods, the magnetic beads are surface enhanced(e.g.:epoxy-coated) magnetic beads. In an embodiment of the methods, thenon-magnetic beads are carboxyl modified beads.

In an embodiment of the methods, the agents are attached to the beads bycovalent binding.

In an embodiment of the methods, optically-active labels are chosen fromthe group of fluorophores and nanocrystals.

In an embodiment of the methods, the sample is a lysate and magneticbeads are separated from the lysate using a magnetic field.

In an embodiment of the methods, the optical signal is quantified byfirst exposing the complexed beads to one or more excitation lightsources, such as, in a non-limiting embodiment, a laser. In anembodiment of the methods, the optical signal is quantified by firstexposing the complexed beads to one or more lasers.

In an embodiment of the methods, the sample is contacted with the firstagent(s) and second agent(s) under conditions which permit capturing tothe primary agents a first protein-second protein complex from thesample.

In an embodiment of the methods, the sample is a lysate and thenon-magnetic beads are separated from the lysate by centrifugation.

In an embodiment of the methods, the cell or tissue lysate is fromprimary isolated cells, lymphoblasts, fibroblasts, cancer cells, a cellline, transfected cells, tissue or blood.

In an embodiment of the methods, quantitatively measured opticalactivity of labeled agents bound to the complex is converted into arelative or an absolute quantitation number of co-binding molecules ineach complex.

In an embodiment of the methods, forward scatter amplitude gain and sidescatter voltage on a flow cytometer are set to register populations ofbead events to on scale, followed by applying an inclusion gate whereselected linear populations of beads form collective clusters containinginterrogation targets can be analyzed in their entirety by flowcytometry.

Also provided is a kit for detecting changes in protein expression incells and for analysis of gene variants, the kit comprising:

-   magnetic beads for immunoprecipitation,-   non-magnetic beads for immunoprecipitation,-   a lysis formulation,-   one or more Proteinase K inhibitors,-   one or more phosphatase inhibitors,-   a coupling buffer,-   nucleic acids recovery elution buffer,-   one or more functional variant assay (FVA) buffers,-   a Western loading buffer,-   one or more optically active labels, and-   instructions for use of the kit.

In an embodiment, the kit further comprises one or more of:

-   a nucleic acid recovery buffer,-   a proteinase inhibitor,-   a fix-permeabilization buffer, and-   one or more primary agents for capturing protein-protein complexes    or protein-nucleic acid complexes.

In an embodiment, the optically active labels are nanoparticles and/orfluorescent dyes.

In an embodiment, primary agents are attached to the beads. In anembodiment, a portion and/or subset of the beads are labeled with anoptically active agent.

Also provided is a method of detecting and analyzing a gene variantbased on a protein-protein interaction, the method comprising:

attaching a first primary agent to the surface of magnetic beads thatare not labeled with an optically active label and attaching a secondprimary agent to the surface of magnetic beads that are labeled with anoptically active label;

attaching a third primary agent to the surface of non-magnetic beadsthat are not labeled with an optically active label and attaching afourth primary agent to the surface of non-magnetic beads that arelabeled with an optically active label, wherein the first, second, thirdand fourth primary agents are different agents and wherein the first,second, third and fourth primary agents are each capable of capturing adistinct protein complex from a cell or tissue lysate;

capturing to the primary agents a protein complex from a cell or tissuelysate, where the protein complex comprises a protein of interest thatis a product of a gene or a gene variant and where the protein ofinterest forms part of a complex with another protein;

probing the protein-protein complex with one or more optically activesecondary agents specific for a member of the complex; wherein the sameone or more optically active labels can be used to label secondaryagents on any of i) the magnetic beads that are not labeled with anoptically active label, ii) the magnetic beads that are labeled with anoptically active label, iii) the non-magnetic beads that are not labeledwith an optically active label, and iv) the non-magnetic beads that arelabeled with an optically active label;

separating protein-magnetic bead complexes from the lysate based onmagnetic properties of the magnetic beads;

separating protein-non-magnetic bead complexes from the lysate based ona physical property of the non-magnetic beads; and

measuring optical activity of optically active-labeled agents on theprotein-bead complexes,

wherein the absence or presence of the optically active label on themagnetic beads is used to distinguish optically active protein complexescaptured by the first and second primary agents, respectively, andwherein the absence or presence of the optically active label on thenon-magnetic beads is used to distinguish optically active proteincomplexes captured by the third and fourth primary agents, respectively.

Also provided is a method of detecting and/or analyzing a gene variantbased on a protein-nucleic acid interaction, the method comprising:

attaching a first primary agent to the surface of magnetic beads thatare not labeled with an optically active label and attaching a secondprimary agent to the surface of magnetic beads that are labeled with anoptically active label;

attaching a third primary agent to the surface of non-magnetic beadsthat are not labeled with an optically active label and attaching afourth primary agent to the surface of non-magnetic beads that arelabeled with an optically active label, wherein the first, second, thirdand fourth primary agents are different agents and wherein the first,second, third and fourth primary agents are each capable of capturing adistinct protein-nucleic acid complex from a cell or tissue lysate;

capturing to the primary agents a protein-nucleic acid complex from acell or tissue lysate, where the protein-nucleic acid complex comprisesa gene or a gene variant nucleic acid sequence;

separating protein-nucleic acid-magnetic bead complexes from the lysatebased on magnetic properties of the magnetic beads;

separating protein-nucleic acid-non-magnetic beads complexes from thelysate based on a physical property of the non-magnetic beads;

digesting proteins on the protein-nucleic acid bead complexes to releasenucleic acids; and

amplifying the released nucleic acids;

wherein the absence or presence of the optically active label on themagnetic beads is used to distinguish optically active protein-nucleiccomplexes captured by the first and second primary agents, respectively,and wherein the absence or presence of the optically active label on thenon-magnetic beads is used to distinguish optically activeprotein-nucleic acid complexes captured by the third and fourth primaryagents, respectively.

The invention provides a method of detecting and/or analyzing a genevariant based on changes of protein-protein interactions, the methodcomprising:

attaching a first primary agent to the surface of magnetic beads thatare not labeled with an optically active label and attaching a secondprimary agent to the surface of magnetic beads that are labeled with anoptically active label;

attaching a third primary agent to the surface of non-magnetic beadsthat are not labeled with an optically active label and attaching afourth primary agent to the surface of non-magnetic beads that arelabeled with an optically active label, wherein the first, second, thirdand fourth primary agents are different agents and wherein the first,second, third and fourth primary agents are each capable of capturing adistinct protein complex from a cell or tissue lysate;

capturing to the primary agents a protein complex from a cell or tissuelysate, where the protein complex comprises one or more proteins ofinterest, where the protein of interest is a product of a gene or a genevariant and where the protein of interest forms a complex with anotherprotein;

probing the protein-protein complex with one or more optically activelabeled secondary agents specific for a member of the complex; whereinthe same one or more optically active labels can be used to labelsecondary agents on any of i) the magnetic beads that are not labeledwith an optically active label, ii) the magnetic beads that are labeledwith an optically active label, iii) the non-magnetic beads that are notlabeled with an optically active label, and iv) the non-magnetic beadsthat are labeled with an optically active label;

separating protein-magnetic bead complexes from the lysate based onmagnetic properties of the magnetic beads;

separating protein-non-magnetic bead complexes from the lysate based ona physical property of the non-magnetic beads; and

measuring optical activity of optically active-labeled agents probed onthe protein-bead complexes,

wherein the absence or presence of the optically active label on themagnetic beads is used to distinguish optically active protein complexescaptured by the first and second primary agents, respectively, and

wherein the absence or presence of the optically active label on thenon-magnetic beads is used to distinguish optically active proteincomplexes captured by the third and fourth primary agents, respectively.

The invention also provides a method of detecting and/or analyzing agene variant based on changes of protein-nucleic acid interactions, themethod comprising:

attaching a first primary agent to the surface of magnetic beads thatare not labeled with an optically active label and attaching a secondprimary agent to the surface of magnetic beads that are labeled with anoptically active label;

attaching a third primary agent to the surface of non-magnetic beadsthat are not labeled with an optically active label and attaching afourth primary agent to the surface of non-magnetic beads that arelabeled with an optically active label, wherein the first, second, thirdand fourth primary agents are different agents and wherein the first,second, third and fourth primary agents are each capable of capturing adistinct protein-nucleic acid complex from a cell or tissue lysate;

capturing to the primary agents a protein-nucleic acid complex from acell or tissue lysate, where the protein-nucleic acid complex comprisesa gene or a gene variant nucleic acid sequence;

separating protein-nucleic acid-magnetic bead complexes from the lysatebased on magnetic properties of the magnetic beads;

separating protein-nucleic acid-non-magnetic bead complexes from thelysate based on a physical property of the non-magnetic beads;

digesting proteins on the protein-nucleic acid complexes to releasenucleic acids; and

amplifying the released nucleic acids;

wherein the absence or presence of the optically active label on themagnetic beads is used to distinguish optically active protein-nucleiccomplexes captured by the first and second primary agents, respectively,and

wherein the absence or presence of the optically active label on thenon-magnetic beads is used to distinguish optically activeprotein-nucleic acid complexes captured by the third and fourth primaryagents, respectively.

In any of the methods disclosed herein, a washing step can be performedbetween contacting and recovering steps in order to remove unwanted orunbound materials.

In any of the methods or kits disclosed herein, the magnetic beads canbe, for example but not limited to, epoxy-coated magnetic beads. In anyof the kits or methods disclosed herein, the non-magnetic beads can be,for example but not limited to, carboxyl modified beads. Examples ofbeads that can be used the methods and kits disclosed herein include,but are not limited to, Dynabeads® M-270, Dynabeads® M-450, CarboxylModified Latex Beads and Dynabeads ClinExVivo Epoxy from InvitrogenCorporation, Carlsbad Calif.

Additional agents can be attached the surface of the beads, andprotein-protein and protein-nucleic acid complexes, to increase thecomplexity of the functional assay.

In any of the methods or kits disclosed herein, the agents can be one ormore of antibodies, monoclonal antibodies, polyclonal antibodies,antibody fragments, F(ab′h fragments, Fab′ fragments, peptides,nucleotides, peptide nucleic acids, and small biological and/or chemicalcompounds. The small compound can have a molecular weight of, forexample but not limited to, 2,000 daltons or less, e.g., 1,000-2,000daltons.

In any of the methods or kits disclosed herein, agents can be attachedto the beads, for example but not limited to by chemical binding, suchas, e.g., covalent binding.

In any of the methods or kits disclosed herein, beads can be labeledwith an optically active label by, for example, using an opticallyactive primary agent. Alternatively, or in addition, beads can belabeled with an optically active label by using an optically activeagent that is different than the primary agent used to capture proteincomplexes or protein-nucleic acid complexes.

In any of the methods or kits disclosed herein, the optically activelabel can be, for example but not limited to, a fluorescent label and/ora nanocrystal (e.g., QDot®).

An optically active label having the same unique wavelength (forexample, color green) can be used to label secondary agents on any of i)the magnetic beads that are not labeled with an optically active label,ii) the magnetic beads that are labeled with an optically active label,iii) the non-magnetic beads that are not labeled with an opticallyactive label, and iv) the non-magnetic beads that are labeled with anoptically active label. This allows for multiplexing of labels toidentify distinct protein-protein and/or protein-nucleic acid complexes.

In any of the methods disclosed herein, the magnetic beads can beseparated from the lysate using a magnetic field. In methods disclosedherein, the non-magnetic beads can be separated from the lysate theirphysical properties, for example but not limited to centrifugation.Alternatively, after the magnetic beads are removed from the lysate,non-magnetic beads can be separated from the lysate by binding (covalentor non-covalent) the non-magnetic beads to secondary magnetic beads andseparating the bound non-magnetic/magnetic bead complex using a magneticfield.

In methods disclosed herein, quantitatively measured optical activity ofoptically active agents bound to the complex can be converted into anabsolute or estimated relative number of co-binding molecules in thecomplex. Optically active beads can be counted by optical readers, forexample but not limited to a flow cytometer and/or plate reader.

In methods disclosed herein, the methods can comprise techniques ofselecting subpopulation of beads with analyte complex by means ofsorting for particular targets of interest based on optical propertiesfor further analysis and/or downstream applications, for example but notlimited to purification of the captured analytes.

In methods disclosed herein, forward scatter amplitude gain and sidescatter voltage on a flow cytometer can be set to register populationsof bead events to on scale, followed by applying an inclusion gate wherelinear selected populations of beads are analyzed so that complexpopulations of the collective clusters containing interrogation targetscan be analyzed in their entirety by flow cytometry.

In methods disclosed herein, captured/purified nucleic acids can beamplified using any conventional method, e.g., sequencing, polymerasechain reaction, multiplex ligation assay, etc.

In methods disclosed herein, the cell or tissue lysate can be from,e.g., primary isolated cells, such as, lymphoblasts, fibroblasts, normalor diseased tissues, cancer cells or any cell lines not limited totransfected cells and from tissues or blood.

With any of the methods or kits disclosed herein, a wild-type gene canbe compared with a gene variant with or without transgene overexpression(exogeneous).

The invention provides a kit for detecting changes in protein expressionin cells and for analysis of gene variants, the kit comprising:

-   -   magnetic beads for immunoprecipitation,    -   non-magnetic beads for immunoprecipitation,    -   a lysis formulation,    -   one or more Proteinase K inhibitors,    -   one or more phosphatase inhibitors,    -   a coupling buffer,    -   nucleic acids recovery elution buffer,    -   one or more (e.g., a set of) functional variant assay (FVA)        buffers,    -   a Western loading buffer.    -   one or more optically active labels, and    -   instructions for use of the kit.

The optically active labels can be, for example but not limited to,nanoparticles and/or fluorescent dyes.

The kit can also include, for example, one or more of a nucleic acidrecovery buffer (CHIP digest buffer), a protease inhibitor, afix-permealization buffer, and one or more primary agents for capturingprotein-protein complexes or protein-nucleic acid complexes.

The kit can include one or more primary agents for capturingprotein-protein complexes or protein-nucleic acid complexes. The primaryagents can be attached to the beads. A portion and/or subset f the beadscan be labeled with an optically active agent. The kit can include, forexample, one or more optically active agents for binding to aprotein-protein complex or to a protein-nucleic acid complex.

The kit and methods disclosed herein allow techniques of selectingsubpopulation of beads with analyte complex by means of sorting forparticular targets of interest for further analysis and/or downstreamapplications, for example but not limited to purification of thecaptured analytes.

Examples of phosphatase inhibitors than can be used include, but are notlimited to, 10× sodium orthovanadate stock and 10× sodium fluoridestock. Examples of Proteinase K inhibitors than can be used include, butare not limited to, 10× Super Proteinase K inhibitor cocktail. Forexample, dissolve Proteinase K inhibitor cocktail (Sigma, cat. no. P2714) in 900 μL distilled H₂O. It also contains other inhibitors, forexample but are not limited to AEBSF (4-(2-aminoethyl)benzenesulfonylfluoride, Sigma, cat. no. A8456), bestatin, aprotinin,Ethylenediaminetetraacetic acid (EDTA), E-64, and leupeptin. Examples ofdetergents that can be used include, but are not limited to, nonionicdetergent: e.g. Triton X-100, NP-40, and digitonin.

The instructions for use of the kit can include any of the instructionsset forth within the present application.

The invention provides kits for the following:

-   1. Identification of the protein-protein interactions;    -   1.1. The kit includes a bar-coded bead system, for example but        not limited to epoxy-modified Dynabeads and CML beads, buffers,        and recommended optically active agents, for example but not        limited to fluorescent dyes (one vial for each conjugate dyes        for attaching to an agent, for example but not limited to target        antibodies). A complement of buffer system for all steps from        fixation, permeabilization and staining of cells, lysis of cells        or tissues, protein, FVA washes and flow analysis.-   2. Protein-nucleic acid interactions;    -   2.1. Gentle elution buffer for active protein-nucleic acid        complex purification, and sample recovery after FVA analysis by        sorting. The kit includes a protein digestion chemistry in the        form of, for example but not limited to, DNase and RNAse-free        proteinase K, Upon performing FVA or Flow sorting, population of        beads can be precipitated by applying magnetic field, and by        physical separation of the second population of beads with        another complex. No wash needed. A kit digestion buffer mix is        added to the barcoded bead systems for 2 hour or overnight        digest to the beads. DNA elution and purification at neutral pH        to enrich captured nucleic acids for applications of MPS        followed after CHIP, allelic discrimination assays, quantitative        PCR expression analysis, RNA IP, RNA CHIP and CHIPs PCR assays.-   3. Cell-based protein expression;    -   3.1. A digital cell western system (DCW) to fix and permeabilize        cells for staining of internal cellular expression of analytes        is designed in the kit. This replaces the need to perform        separate Western blot prior to IP, or standard westerns.-   4. Protein modifications;    -   4.1. With the available of antibodies for modified proteins        (phosphorylation, acetylation, prenylation, ubiquitination), and        other antibodies or agents that can detect modifications of        analyte, the kit is design to incorporate these agents as part        of the unified assay read out.-   5. Concurrent immunoprecipitation (IP)-Western;    -   5.1. The recovered beads after FVA analysis can be treated with        the kit Western elution buffer containing Sodium dodecyl sulfate        (SDS) and dye designed for downstream traditional Western blot.        This reduces technical variations when comparing experimental        readout data from the same sample.-   6. Generation of reference controls using the FVA kit conjugated    antibodies;    -   6.1. The conjugation buffer is adapted to perform conjugation of        the freshly made optically active, for example but not limited        to fluorescently labeled, antibodies to the beads rapidly. These        are the antibodies that will be used to detect the FVA analytes.        This increases consistency as the same host, and recognition        epitope as in the detection antibodies are used.

The invention provides methods that include:

-   1. Methods to identify the protein-protein interactions;    -   1.1. Bait antibody is conjugated on to the surface of the epoxy        modified beads, a set of optically active multi-analyte        antibodies can be used to detect the bound proteins using        methods of FVA. This allows identification of proximal binding        targets within one complex. FVA allows quantitative measurement        of up to various targets simultaneously. With the use of FVA,        selection of particular variant interactions can be selected by        gating inclusion/exclusion sorter, and be eluted for further        assays and other analysis that is otherwise not possible by        conventional means. The kit includes steps to use these same        analyte agents, such as for example antibodies, to generate        optical standards that can be used to accurately establish the        threshold of detection based on its optical standard        intensities.-   2. Methods to identify protein-nucleic acid interactions;    -   2.1. A simplified workflow that includes nucleic acid enrichment        after FVA by elution of protein-nucleic acid complex, digest,        release of nucleic acids and enrichment methods, for example but        not limited to ligation of adaptamers to the fragments of        captured nucleic acids to perform digital emulsion or standard        PCR steps to assess the bound regions to the target sequences        and their variants using optical scanners, for example        sequencer, flow cytometer or plate reader. The target sequence        and its variant forms (single nucleotide polymorphisms,        insertions and deletions, and structural rearrangements) can be        measured by using optically active probes annealed to the        amplified targets on the beads. In addition, the kit allows        novel methods to identify binding sites of the RNA with the        interacting proteins in its native form, adapted to a high        throughput and high content analysis format.-   3. Cell-based protein expression (DCW);    -   3.1. Prior to lysing of the cells for FVA, a quick assessment of        the expression levels of the analyte can be performed by        sampling using DCW to fix, permealize, and immunostain the        cells. Multiple protein targets can be measured simultaneously.        Each detention data point is considered to be one cell western,        and aggregate of cells generates a digital value of the        cumulative data points, as digital cell western.-   4. Protein modifications;    -   4.1. DCW methodology is optimized to incorporate available        analyte antibodies to detect protein modifications, where the        antibodies for specific post translational modification can be        conjugated with the optically active agent.-   5. Concurrent immunoprecipitate (IP)-Western;    -   5.1. Beads that are retained after FVA analysis can be placed        into denaturing western loading buffer in the kit to perform        standard IP-western blot.-   6. Generation of reference controls using the optically active    conjugated antibodies;    -   6.1. In traditional protocols, prior to performing flow        cytometry analysis, a set of commercial bead-based reference        dyes is used to create fluorescence standards; drawbacks are        commonly acknowledged that the host epitope of antibodies for        the analyte is greatly different from the references. The FVA        reference control methodology resolve this issue by using the        same conjugated antibodies as calibration controls by        conjugating them on to the bead systems.

The interaction of the protein of interest (“bait”) or variant proteinwith its associated targets, and multiple gene variant complexes can beexamined simultaneously. This has the effect of looking not only at amajor effect of a variant in the bait gene(s), but also at the epistaticinteractions of a variant in second gene(s) with the bait products ofother genes in the complex.

By altering the gating and an inclusion principle of counting apopulation of subsets of beads previously missed, the present inventiongreatly improves sensitivity by at least 10-fold. By improving thesensitivity, the amount of starting material can be reducedsignificantly and by using a bar-coded bead system, the binding time canbe reduced dramatically from 24 hours (overnight) to 5 hours formultigene functional assay.

By gating and sorting for analytes of interests, the present inventionallows for selection and enrichment of target proteins or nucleic acidsthat would simply not be possible using current technologies.

The kit provides all the steps and chemical reagents necessary toperform a DCW assay, with addition of one initial startup step, i.e.,permeabilize the cells prior to the FVA standard protocol.

As used herein, one form of “bar coding” means to identify differentprotein-protein complexes or different protein-nucleic acid complexes onbeads with different optically active labels (“vertical bar coding”).Another form of bar-coding means to identify different analytes on themagnetic beads and/or non-magnetic beads using optically active labels(“horizontal bar coding”). For example, but not limited to, for 7different fluorescent labels, one label can be used to identify aportion and/or subset of magnetic or non-magnetic beads and theremaining 6 labels can be used to label different protein-proteincomplexes or different protein-nucleic acid complexes attached to thebeads.

Examples of apparatus that can be used with the disclosed methodsinclude, but are not limited to, a 7-color flow laser machine or a13-color flow laser.

The methods disclosed herein allow unprecedented speed to study effectsof mutations and offers high sensitivity to detect mutant proteinactivities, such as changes in protein-protein interactions,modifications and mutant protein co-localization into the nucleus.Traditionally, a transgene is needed for each interrogated mutant,whereas the present method uses cells isolated from actual subjectbearing the mutation, hence there is no need to create any transgeneprior to the FVA assay. In an embodiment of the methods, no transgene isinserted into the cells of the sample. Additionally, conventionalmethods struggle to detect endogenous protein accurately, but theincreased sensitivity of the present methods allow accurate detection ofthe mutant protein and of its activities. This changes the paradigm ofhaving to genetically engineer mutant cells to study its biology, tosimply using the isolated subject cells for various assays described. Inan embodiment of the methods, no the cells of the sample have not beenpreviously genetically engineered. The methods herein allow effectivecost reduction for large-scaled studies of mutation.

This invention will be better understood from the Experimental Detailsthat follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims that followthereafter.

EXPERIMENTAL DETAILS Example A—Methods Introduction

The dynamic range of Co-IP Western blots can be improved by flowcytometry, a direct counting function (13). Using this method,immunoprecipitating antibodies, known as ‘bait’, are coupled covalentlyto polystyrene beads whose low autofluorescence is suitable for flowcytometry detection. Once coupled, the antibodies on beads willimmunoprecipitate a specific protein complex in cell lysates. Thesecomplexes can then be probed with a panel of fluorescently taggedsecondary antibodies to quantify the binding of the interacting partnersby quantitatively count the bound beads on a flow cytometer at highspeed. Here, this method has been applied to probe a clinical case studyusing wild-type B lymphoblastoid cell lines or those derived frompatients with MAP3K1 mutations who have 46,XY gonadal dysgenesis (14).

The present studies show altered binding of interacting proteins thatcould influence downstream signaling in testis or ovarian-determiningpathways. Because B cell lines express a large repertoire of proteincomplexes and can be derived from patients with presumed geneticdisorders in a non-invasive manner, they represent a readily availableresource for screening variants of uncertain phenotypic significance.

Materials and Methods

Cell culture and reagents. Three MAP3K1 mutation-bearing Epstein-Barrvirus B-lymphoblastoid cells lines (c.634-8A, p.Leu189Arg, andp.Leu189Pro) from individuals with 46,XY complete gonadal dysgenesis anda wild-type cell line were derived, as described previously (14). Thesecell lines were grown in RPMI medium (Invitrogen A2780), supplementedwith 15% (v/v) FBS Defined Grade, 50 units/mi penicillin, and 50 g/mlstreptomycin at 37° C. with 5% CO₂. The antibodies included MAP3K1rabbit polyclonal antibody (Lifetech #51-340), RHOA rabbit polyclonalantibody (Abcam #ab66124). MAP3K4 mouse monoclonal (Abcam #55669), IRDye800 CW Goat anti-Mouse (Licor #926-32210), and IRDye 680LT Goatanti-Rabbit IgG (Licor #926-68021). Western blotting analysis wasperformed as described previously (14).

FVA on cultured lymphoblastoid cells. FVA was performed with thefollowing modifications optimized for lymphoblastoid cell lysates. Thebait antibody was conjugated onto epoxy modified beads at concentrationsof 30 μg of antibody per mg of 5 micron epoxy beads, conjugation isachieved using the coupling buffers in the kit. The Proteinase K andphosphatase inhibitors at 2 or 4× concentration were added to the celllysates during lysis. Antibody conjugated epoxy beads were added to thelysates to incubate for 5 hours or overnight at 4° C. Alexa 488 and 647dyes were coupled to the secondary antibodies using labeling kits(Lifetech #A20181 and A20186, respectively). The secondary antibodieswere added to the lysate after 2 washes of the previous incubation, andwere incubated at room temperature (20° C.) for 30 minutes with 2additional washes following to remove unbound antibodies. Flow cytometrywas performed on a BD FacsCantoII with 96-well, high-throughputcapabilities. The counting of bead fluorescence occurrences, ‘events’,was set at 10,000-25,000 gated events setting from the previouslypublished 2500 events (13). Note that ‘gating’ refers to the combinationof fluorescence and scatter values that are counted.

Results

The intrinsic fluorescence of the kit beads was low and did notinterfere with subsequent analysis of binding fluorochromes. Thefluorescence of the Alexa 488 and 647 dyes on antibody-conjugated epoxybeads was used, formulated using the kit coupling buffers (see protocolstep 2 in “Preparation before starting”) as reference control forcalibrating the flow cytometer gate sensor to exclude background noiseand to gauge dye detection and recognition fidelity. Positive controlsfrom binding of either MAP3K1 or RHOA complexes to the beads could, inturn, be recognized by the labeled MAP3K1 and RHOAantibodies—demonstrating the specificity of these IP pull-downreactions. Western blot analysis was performed to confirmed that theexpression of wild-type and mutant MAP3K1 proteins and that the inputamounts of MAP3K prior to IP were consistent (FIGS. 4C and 6A).Previously, Western blot analysis showed that the input amounts of RHOAprior to IP were consistent (14). The IP Western from the MAP3K1pull-down of MAP3K4, and the RHOA pulldown of MAP3K1 in the aggregateshowed dramatic increases in binding among mutant samples as quantifiedby densitometry analysis using Licor Software 3.0 (FIGS. 6B and 6C).When applied to the analysis of the patient and control samples tomeasure protein interactions, the binding of MAP3K4 was shown by FVA tobe increased 3-fold in these cell lines that contain any of threeendogenous mutant MAP3K1 genes (C.654-8A, p.Leu189Pro and p.Leu189Arg)compared to wild-type cell lines (FIG. 4). The results observed with FVAwere confirmed by conventional IP Western blots of samples eluted fromthe epoxy beads, although the binding appeared to be increased 2-fold onaverage among all mutants, similar results were observed in previoustraditional IP Western. Accordingly, it was confirmed that the increasesobserved were not the result of unequal loading nor increased expressionof MAP3K1, as illustrated by actin Western as loading control and MAP3K1input prior to IP (FIG. 6B).

These methods were applied to the reverse immunoprecipitation method inwhich the ‘bait’ was RHOA, a MAP3K1-interacting protein to showconsistency, and binding of wild-type or mutant MAP3K1 was measured byflow cytometry (FIG. 5). In this case, the binding of the mutant MAP3K1proteins was increased 2 to 4-fold in cells containing mutant comparedto wild-type MAP3K1. The results observed with FVA were confirmedconsistent, and by conventional immunoprecipitation Western blots ofsamples eluted from the epoxy beads, notably the binding from all mutantcells appeared to be increased 2.5-fold compared to wild-type (FIGS. 5Cand 6C). This discordance has been accounted for by the limited dynamicrange of conventional Western blots compared to the sensitivity of flowcytometry.

DISCUSSION

DNA sequencing of candidate genes or whole exomes on a diagnostic orinvestigational basis will yield a plethora of variants whose potentialphenotypic roles cannot be readily demonstrated by prediction programs,SNP databases nor conventional genetic analysis. Many variants mayproduce phenotypic changes in the encoded proteins by affecting thequantity, post-translational modification or protein interactions. Thepresent studies establish the application of the method of FVA todemonstrate that known protein interactions are altered in the Blymphoblastoid cells of patients with 46,XY gonadal dysgenesis arisingfrom mutations in the MAP3K1 gene. This method can be scaled readily totest multiple interactions for many variants simultaneously fromavailable tissues as well as quantify the effects of variants on proteinaccumulation and post-translational modification, thus functionalscreening of gene variants for phenotypic effects is made possible bythis efficient and low-cost FVA.

Here, it was shown that the effects of missense and in-frame splicing(with insertion of 2 amino acids) mutations in the amino third of theMAP3K1 gene result in increased binding of MAP3K4. This might arisethrough interactions with their shared binding partner, AXIN1 (15-16).Both MAP kinases compete for AXIN binding, albeit at different sites(15), and the presence of these MAP3K1 mutations may alleviate thiscompetition. Unlike MAP3K1, MAP3K4 is an essential testis determininggene. Homozygous loss of function alleles in mouse Map3k4 lead todisrupted testis development in mice from failure to support corddevelopment (17), whereas knockout of the mouse Map3k1 gene does not(18). Reduction of MAP3K4 protein either from genetic knockout or fromsequestration in MAP3K1-MAP3K4 complexes may have functionally similareffects. Increased binding of MAP3K4 to MAP3K1 complexes as shown by FVAmay affect downstream WNT targets through AXIN1. AXIN1, an inhibitor ofthe WNT signaling pathway, interacts with β-catenin to reduce itsprotein abundance (16, 19). This results in an increase and/orstabilization of β-catenin, an effect that is known to causemale-to-female sex reversal in the XY gonad—in part by reducing SOX9expression (20).

The present studies have also shown accurate detection of changes ofbinding partners to MAP3K1 in all three mutant MAP3K1 by FVA casesconsistent with previous studies using traditional methods but at ahigher fidelity (21).

These approaches of FVA, either with or without immunoprecipitation, canbe applied to test other candidate gene variant expressions andfunctional implications. In the process, FVA can measure the generaleffects that a variant might have for a protein and its regulation ofdownstream targets. High-content measurements such as alteration of itsexpression and effects on the expression and accumulation of otherdownstream proteins in the cell, alteration of the post-translationalmodification of the variant protein, such as phosphorylation, oralteration of the variant and/or wild-type protein with its co-factors.Typically a handful of variants are selected for biological assays bygenetic manipulation in cells or animals to show that a newly identifiedvariant is a mutation. i.e. has a phenotypic effect. Previously, it hasbeen shown that the effects on accumulation and post-translationalmodification can be measured reliably by flow cytometry in research andclinical applications (13, 27). The improved methods of FVA and kitpresented here have been tested on human in-vitro studies using primarycells isolated from the subjects, and compared to standard geneticmanipulated human cell lines to demonstrate for the use of FVA ashigh-throughput screening for the effects of genetic variants on bindingof partners, at proteomic level (functional assay) that are readilyadaptable for single-tube, 96- or 384-well approach. Thus, functionalanalysis of multiple variants and multiple binding partners in a singleexperiment in one or two days can be performed reliably and costeffectively. Moreover, comparison of previous IP-Western densitometryresults with this invention FVA shows a greater sensitivity using onlyone-fifth the amount of starting material compared to traditionalmethods. The multiple binding partners can be tested in the same tube,because analytes using optically active antibodies each with differentemission wavelengths can be measured simultaneously (27). Furthermore,the method can test not only interactions with binding partner, but alsothe quantification of the bound protein itself and itspost-translationally modified forms, such as the phosphorylation statusof the MAP kinase (28). The present methods and kits have beenextensively tested in several other clinical genetic studies whereB-lymphoblastoid cells isolated and immortalized from patients wereused; these type of blood cells are commonly use in traditional geneticscreening. As stated previously, the method was also tested in NeuronalTeratocarcinoma 2 (NT2) cells, a standard cell line routinely used inresearch laboratories to study gene variant effects by geneticmanipulations.

In conclusion, this method proves to have clinical diagnostic value, anon-invasive, and cost effective test suitable for virtually anyvariants that are identified by massive parallel sequencing andgenome-wide association studies (GWAS). In addition, the methods can beused to survey and identify the genetic heterogeneity of populations ofcells. The methods can also be used to investigate epistaticinteractions between proteins, wherein one protein is the distinct firstprotein of the method and the other protein is a distinct secondprotein.

Example B—Kits Introduction

Protein expression is deemed to be a gold standard for measuring changesin gene activities. Many variants identified through sequencing ormutant characterization may produce phenotypic changes in the encodedproteins by affecting the quantity, post-translational modification orprotein interactions. Indeed, the frequency of rare sequence variants isproving to be far higher than previously thought. Complex proteininteractions play crucial roles in virtually all cellular processes.Traditionally, such protein-protein interactions were studied viaco-immunoprecipitation. However, this method is laborious and are onlyuseful for small number of targets, require large amount of biomaterialto start, not clinically sensitive and costly as each target must bemeasured independently in separate Western blots. Analysis ofco-immunoprecipitation of protein complexes using FVA provides asensitive rapid method to measure multiples of these interactions intheir native state. This kit provides all the essential and optimizedreagents to perform the assay along with a validated ‘bait’ antibody.First, target bait antibodies are covalently coupled to the bar-codedbead system in the kit. These antibody-coupled beads are used as baitfor protein lysates. Finally, the pulled-down protein complexes on thebeads are separated based on their properties and probed with opticallyactive agent-labeled antibodies specific for interaction partners andmeasurement of a quantitative fluorescence are converted into quantityor numbers of co-binding molecules in a complex. FVA represents a robusttechnique to assess native protein-protein interactions rapidly withvery small amount of biomaterials. This kit includes, for example butnot limited to, seven optically active agents with minimal spectraloverlap to measure multiple targets simultaneously with a standardthree-laser flow cytometer, with methods that can be performed in justunder one day, and alternative protocol allows for a two-day assay ifneeded.

Absolute Quantitative Flow Cytometry

Optical activity data are often presented on a relative scale witharbitrary units because it is inherently semi-quantitative bytraditional means. This kit allows both measurements of semi andabsolute quantification. This kit provides a set of standard opticallyactive reference beads created using the same antibodies selected foreach tests to generated standard curve, where optical activity valuescorrespond to known numbers of molecules. Using this standard curve, onthe same flow cytometer settings, absolute quantitation can be measuredby translating the measured relative optical activity values from theFVA into numbers of target molecule per IP bead.

Kit Components

Kit Components can Include:

Immunoprecipitate Beads (Store at 4° C.)

Examples include, but are not limited to, polystyrene-epoxy paramagnetic(Dynabeads) beads (5 μM) in storage buffer, carboxylate-modifiedpolystyrene surface latex (CML) beads (5 μm) in storage buffer, 10 ml ofFlow grade neutral buffered salt solution, pH 7.4. Examples of buffersinclude Phosphate Buffered Saline, Phosphate Buffeted KCL,Trisaminomethane (Tris)-buffered ammonium chloride.

Coupling Buffers (Store at 25° C.)

Coupling buffers at pH range of 4-7 with use of agents, for example butnot limited to, 2-[N-morpholino]ethanesulfonic acid (MES)) that allowcovalent coupling of the agents on to the beads, chelating agents, forexample but not limited to EDTA, and activating compounds, for examplebut not limited to 1-ethyl-3-(3-dimethylaminopropyl) Carbodiimide HCl(EDAC) are added as well.

Fix-Permealization Buffer (DCW Buffer) (Store at 4° C.)

A kit for fix and permeabilizing cells comprising but not limited to (a)an isotonic or hypertonic fixing agent at pH 4 to 7 containing, forexample but not limited to an aliphatic aldehyde or alcohol(glutaraldehyde, para- or formaldehyde, alcohol), which is present in aconcentration of at least 5%, (b) a permeabilizing agent, for examplebut not limited to at pH 4 to 8 containing a blocking agent (Bovineserum albumin, BSA) and selected group consisting of Zwitterionic andthe use of ionic detergent (e.g., Sodium dodecyl sulfate, SDS).

Inhibitor Cocktail (Store at −20° C.) Phosphatase Inhibitor Cocktail

A kit containing phosphatase inhibitor that inhibits phosphatases.Examples of phosphatase inhibitors than can be used include, but are notlimited to, sodium orthovanadate and sodium fluoride, and contain apreservative such as Sodium Azide.

Proteinase K Inhibitor Cocktail

A kit containing Proteinase K inhibitor that inhibits Proteinase Ksincluding for example but not limited to chymotrypsin, kallikrein,plasmin, thrombin, and trypsin. Examples of inhibitors include, but arenot limited to, phenylmethanesulfonyl fluoride (PMSF) or4-(2-Aminoethyl)benzenesulfonyl fluoride (AEBSF), and contain apreservative such as Sodium Azide.

Universal Buffer (Blocking and Storage)

A buffer containing, for example but not limited to, blocking agent(BSA), host serums (Rabbit, mouse, and/or goat serum), preservative(Sodium Azide). Formulations include isotonic and hypertonic saline,buffering agent and salts, examples include but not limited to Phosphatebuffer saline (PBS), Potassium chloride (KCL), Monopotassium phosphate(KH₂PO₄), Sodium chloride (NaCl), Disodium hydrogen phosphate (Na2HPO4)and Tris.

After-IP Buffer

A buffer containing, for example but not limited to, Proteinase Kinhibitors (PMSF or AEBSF), preservative (Sodium Azide), isotonic andhypertonic saline, buffering salts, examples include but not limited toSodium chloride (NaCl).

FVA Buffer

A buffer containing, for example but not limited to, blocking agent(BSA), host serums (Rabbit, mouse, and/or goat serum), preservative(Sodium Azide), isotonic and hypertonic saline, buffering agent andsalts, examples include but not limited to Phosphate buffer saline(PBS), Potassium chloride (KCL) or Sodium chloride (NaCl) and Tris.

Lysis Buffer

A buffer containing, for example but not limited to, a buffering agent(Tris), Proteinase K inhibitors (PMSF or AEBSF), phosphatase inhibitors(Sodium Orthovanadate, Sodium Fluoride), a chelating agent (EDTA),contains hypertonic salt, examples include but not limited to Potassiumchloride (KCL) and/or Sodium chloride (NaCl), non-ionic detergent(Digitonin) or ionic detergent (SDS).

Elution Buffer (Store at 4° C.)

A buffer containing, for example but not limited to, blocking agent(BSA), Proteinase K inhibitors (PMSF or AEBSF), phosphatase inhibitors(Sodium Orthovanadate, Sodium Fluoride), a chelating agent (EDTA), apreservative (Sodium Azide), contains hypertonic salt, examples includebut not limited to Potassium chloride (KCL) and/or Sodium chloride(NaCl), a buffering agent (Trisaminomethane (Tris)), non-ionic detergent(Digitonin) or ionic detergent (SDS, or Triton X-100).

Nuclear Enrichment Buffer (Store at 4° C.)

To remove the cytoplasmic compartment of the cells with or withoutfixation (DCW buffer), resuspend cell pellet in NP40 with Tween 20 lysisbuffer: 30 mM Tris/Hepes, pH 8.0, (0-50 mM) NaCl, 3 mM EDTA, 1.5 mMphenylmethylsulfonyl, fluoride (PMSF or equivalent agent), 1% Tween 20and 1% NP40. Leave on ice for 15 min with pipetting for 5 times, thenpellet the nuclei at 2000-6000 g for 3 min at +4° C. Collect or decantthe supernatant (cytoplasm fraction) supplemented with 300 mM NaCl ifneeded for FVA analysis. Resuspend the nuclear pellet in 100 ul ofUniversal buffer containing host IgG and store 24 hours in 4° C. priorto assay, add 120 ul of methanol if long term storage is desired.

Western Loading Buffer

A buffer containing, for example but not limited to, ionic detergent(SDS, or Triton X-100), a buffering agent (Tris), coloring agent(Bromophenol blue), a hygroscopic simple polyol compound (glycerol) anda small-molecule redox reagent such as Cleland's reagent, exampleinclude but not limited to Dithiothreitol (DTT) or dithioerythritol(DTE).

Nucleic Acids Recovery Elution Buffer Mix

A buffer containing, for example but not limited to, protein digestingagent (proteinase K), a chelating agent (EDTA), isotonic and hypertonicsaline, detergent buffering agent and salts, examples include but notlimited to Phosphate buffer saline (PBS), Potassium chloride (KCL),potassium phosphate (KHPO₄), Calcium chloride (CaCl), Glycine, Tris.

Protease Inhibitor Cocktail

A buffer containing, for example but not limited to, proteinaseinhibitors PMSF and Diisopropyl phosphorofluoridate (DPF)) and achelating agent (Ethylene glycol tetraacetic acid, (EGTA)).

Preparation Before Starting

Coupling of bait antibodies to beads. Pipette 20 μL (18×10⁶) Dynabeadsinto a 1.5-mL microcentrifuge tube. Wash the beads 2 times in 700 μLBuffer C1, magnetize the tube and remove supernatant after each wash.Then, resuspend the bead pellet in 25 μL Buffer C1; then transfer allinto provided C2 tube (to activate the coupling group on the beads). Mixgently on an orbital shaker for 15 min at room temperature (25° C.).Repeat with CML beads.

Wash the activated beads 2 times in 700 μL Flow PBS, magnetize the tubeand remove supernatant after each wash. Resuspend the activated beads in50 μL Flow PBS. Add 50 μL of the bait antibody (0.2-1.0 mg/mL stockconcentration) to the activated bead mix. Mix for 2 hours at 25° C. byplacing the tube on a vibrating shaker or taped to a standard vortexerat low shake setting. This will ensure sufficient mixing to preventsettling of the beads. Overnight incubation is not necessary as this kitis optimized for rapid coupling.

Wash Ab coupled beads 2 times in 700 μL PBS, magnetize the tube forDynabeads, and remove supernatant after each wash. Place the CML beadsfor 5 minutes of centrifugation at 5000 g. Resuspend the coupled beadsin 100 μL Universal Buffer. These can be stored at 4° C. for at leastone year. Resuspend the beads well before use to ensure consistency foreach experiment.

Optically active agent labeling on antibodies and optical referencecontrol preparation. Up to 150 μg of antibody per reaction can be set upin the provided glass vials. Antibody to dye ratio is 1:1. Totalreaction volume optimally should be at 110 μL finally.

1) Add 10 μL Modifier into the 100 μL of antibodies, mix gently.

2) Pipette all the mix into the lyophilized dye in the glass vial, mixand incubate for 3 hours to overnight in dark at room temperature, 25°C.

3) Add 1 μL of quencher for every 10 μL antibody used. The conjugate canbe used after 30 minutes of quenching.

For optical reference control preparation, follow the entire step of“Coupling of bait antibodies to beads” with the conjugated antibodies.

Performing Digital-cell Western (DCW). Count, and pellet 1×10⁶ cells foreach sample to perform DCW. 16% formaldehyde is added directly into theculture medium, please note final formaldehyde concentration should beat 1.5% and incubated the cells for 10 min at 25° C. or room temperature(RT). Then, pellet the cells (use dissociation media for adherent cells)by low speed centrifugation. Resuspend the cell pellet by vortexing in500 μl ice-cold Methanol and incubated on ice for 5 minutes. Cells canbe stored at −80° C. for long term with minimum degradation.

To perform staining for target protein expression, cells should bewashed twice in 500 μl cold Universal buffer then resuspended with thisbuffer at 50 μl. It is recommended to test fidelity of antibodies, butstandard guidelines is approximately 50 ng of optically active labeledantibodies should be added and incubated for 30 min at 4° C. Then washthe cells twice with 500 μl cold Universal buffer. Finally, samples wereresuspended in 150 μl FVA Buffer and analyzed by flow cytometer (asnoted in Performing FVA Scan)

Performing FVA complex capture. While lysis method and optimal lysisconditions can vary in some cases of transient protein-proteininteractions being investigated, the lysis buffer provided in this kitis designed to meet most applications and is suitable in many cases.Each lysis buffer is sufficient to perform 10 IPs, and contain mostnecessary inhibitors. Alternatively, other lysis buffers in general arecompatible to be used with this kit.

Lysis of 30×10⁶ cells in 100 μL fresh Lysis buffer in a 1.5-mLmicrocentrifuge tube for 10-20 minutes on ice. Scale the lysis volume asneeded. Two sonication pulses into the lysate are highly recommended formost applications. Cell debris and nuclei can be removed by briefcentrifuge of the lysate at 5000 g for 2 minutes at 4° C. Keep thesupernatant and discard the pellet. Add 1×10⁵ of the coupled beads tothe lysate, (recommend using chimney-bottom 96 well plate for thisstep). Use 50 μL of the lysate to perform FVA for each sample. Thelowest volume can be at 5 μL. Place on a vertical rotating wheel 4 hoursin a cold room, alternatively, overnight incubation in cold room can beperformed. Ensure proper mixing to prevent bead settling.

Probing of bead-captured protein with optically active-labeledantibodies. Wash the IP beads two times in 500 μL ice-cold FVA Buffer,magnetize and remove supernatant and transfer it into a second vessel(tubes or plates). Centrifuge for 5 minutes at 5000 g. and removesupernatant, the pellet is the CML-analyte complex, and perform twowashes for Dynabeads and CML beads, magnetize and centrifugerespectively and discard supernatant after each wash. Resuspend thebeads in 20 μL FVA buffer. Add optically active-labeled antibodies tothe samples. Check with vendor's recommendation on antibodyconcentration use. In most applications, add 0.5 μL of stock antibodysolution (at 0.2-1.0 mg/mL) per tube or well, and incubate for 35minutes on ice on an orbital shaker. Wash probed samples two times in500 μL ice-cold FVA buffer, magnetize and remove supernatant after eachwash. Resuspend the beads in 200 μL FVA buffer per sample. Samples arenow ready for FVA scan and analysis.

Performing FVA Scan

The supplied beads in this kit are about 5 μm in diameter, approximatelyhalf the diameter of a typical lymphocyte. Set forward Scatter (FSC) ampgain to 480 and the side scatter (SSC) voltage to 550 to register thepopulation of bead events to on scale. Both Dynabeads and CML beadsshould form distinct clusters/population in a linear fashion, place thegate circle on all distinct populations of beads. The settings and gateshould be adjusted to include only beads populations not cell debriswhich are typically seen below the bead populations.

Use the default collection criterion, which for most applications is10,000-25,000 acquisition events. Adjustment of this criterion to higheracquisition events may be needed for rare target. The staining of thebeads normally produces distinct mode fluorescence intensity (MoFI) whenvisualized on a histogram mode, change to Standard “log mode” for theuse in fluorescence channel(s) detection.

Prepare and use unlabeled, unconjugated beads as the first sample to setthe negative control photomultiplier tube (PMT) voltage. Optionally,bright positive control for the second sample, such as beads withoptically active-conjugated antibodies (See Preparation beforestarting). Initially, a run of internal optical standards is necessary,for each flow cytometer used. This will create a spectral compensationprofile that can be used for subsequent and future FVA scans. The flowcytometer is now ready to acquire the FVA from the probed samples.

Performing Protein-Nucleic Acid Enrichment

Upon completion of flow sorting, population of beads can be precipitatedby applying magnetic field for Dynabeads and transfer the supernatant toa new tube or plate and centrifuge at 5000 g for 5 minutes to pellet theCML beads then discard the supernatant. No wash needed. Heat the beadsto 90° C. for 15 minutes, cool on ice for 5 minutes then add 50 μL of 1×Nucleic acids recovery buffer mix to the captured beads and incubate at55° C. for 2 hour. Apply magnetic field on the Dynabeads digested mixand centrifuge at 5000 g for the CML digested mix, and transfer thesupernatant into respective new tubes/plates. Add 5 μL of 10× Proteinaseinhibitor to the supernatant. Optionally, standard DNA purification canbe performed after this step but the supernatant is suitable for directuse in applications of MPS, allelic discrimination assays, quantitativePCR expression analysis, and RNA/CHIP PCR assays.

Example C—Nuclear Assay—Localization

BRCA1 expression and localization were analyzed in isolated primaryB-Lymphoblastoid cells from individuals with BRCA1 mutations. The assayswere performed as randomized, anonymized samples. The cells were treatedwith Etoposide and UV radiation to cause double-stranded DNA breaks.About 2 million cells were formalin-fixed and methanol permealized. Eachexperiment included three biological and three technical repeats. Thecytoplasmic fraction was removed by hypotonic salt lysis method followedby nucleus enrichment by centrifugation. Flow staining hydration bufferwas used overnight at 4° C. to increase the nucleus ball diameter from2-3 μm to above 5-7 μm, followed by nuclear staining withoptically-labeled primary antibodies. The results demonstrated thatafter treatment with Etoposide and UV radiation (FIG. 7(A)) or the X-Raymimetic drug, Bleomycin (FIG. 7(B)), the localization of BRCA 1 tonuclear foci was markedly lower among mutant samples compared to normalsamples. The samples were compared to IgG (i.e., negative) controls Thebead internal autofluorescence and selected size events were alsocompared. The BRCA1 relative fluorescence intensity was normalized bydividing with the specific events to the total gated events. An inversecorrelation (P<0.0001) was observed between BRCA1 localization/stainingin the nuclear/pathogenicity upon DNA damage drug treatment. All celllines with mutations showed significantly lower staining, whereas normalcell lines demonstrated strong nuclear staining. The staining intensitydenotes BRCA1 localization into the nucleus for DNA double strandedrepair (DSB). The mutations in BRCA1 abrogated this localization andrepair mechanism.

The nuclear localization kit, and method herein, have been developed forthe preparation of enrichment of nuclear, whole-cell and cytoplasmicextracts from cells or tissue. This kit provides method that is simple,fast and effective to measure protein activities within cytoplasmicand/or nuclear compartments of the cell. The nuclear localization kitcan be used to prepare intact nuclear balls (cellular nuclei) to monitortumor suppressor genes activities in whole intact cell and intactcellular nucleus. The nuclear compartment enrichment collected by thiskit can be used for a variety of standard protocols besides PrCo-IP,NACo-IP, FVA, including electrophoretic mobility shift assay (EMSA), DNAfootprinting, Western blotting and preparative purification of nuclearproteins.

Each kit provides reagents for direct lysis of cells. First, the cellsare collected in ice-cold PBS in the presence of phosphatase inhibitorsor fixation (e.g. by 2% paraformaldehyde) to limit further proteinmodifications (expression, proteolysis, dephosphorylation, etc.). Then,the cells are resuspended in hypotonic lysis buffer and strong detergentadded which causes breakage of the cells resulting in leakage ofcytoplasmic proteins into the supernatant. After collection of thecytoplasmic fraction, the nuclei are fixed-permeabilized with, e.g., amethanol-based storage buffer.

To prepare whole-cell extracts, cells are collected in thePBS/phosphatase Inhibitors solution and lysed in the lysis buffer.Solubilized proteins are separated from the cell debris bycentrifugation. The protein concentration of the cell extract can benormalized by counting number of live cells during cell collection, noprotein quantification necessary. Optionally, a Bradford assay can beused at this step. The method or kit can be used to obtain nuclear,cytoplasmic or whole-cell extract from cells or from tissue.

Successful extraction has been performed with NT2/D1, PC3, LNCaP, DU145,and B-lymphoblastoid cells. In an embodiment, the kit used comprises thecomponents as follows:

-   Lysis Buffer AM1 (e.g. 10 ml)-   1 M Dithiothreitol (DTT) (e.g. 500 μl)-   Protease Inhibitor mix (e.g. 500 μl)-   10×PBS (e.g. 4×100 ml)-   Phosphatase Inhibitors (e.g. 4×50 ml)-   10× Hypotonic Buffer (e.g. 50 ml)-   Detergent (NP40 mix) (e.g. 5 ml).

Protocol Examples

The following exemplary protocol is based on samples of approximately20×10⁶ cells, which corresponds to 10 wells on a 96-well plate. Eachwell is one reaction. Prepare PBS/phosphatase inhibitors, hypotonicbuffer and total lysis buffer. Place buffers and any tubes needed on icebefore beginning assay.

Step 1: Cell Collection

-   1. Aspirate media out of culture vessel. Wash with 5 ml ice-cold    PBS/Phosphatase Inhibitors. Aspirate solution out and add 3 ml    ice-cold PBS/Phosphatase Inhibitors.-   2. Remove cells from flask/dish by gently scraping with cell lifter    for adherent cells. Transfer cells to a sterile 50 ml conical tube.-   3. Centrifuge cell suspension for 5 minutes at 500 rpm in a    centrifuge pre-cooled at 4° C.-   4. Discard supernatant. Keep cell pellet on ice.

Step 2: Cytoplasmic Fraction Collection

-   1. Gently resuspend cells in 5 ml 1× Hypotonic Buffer with 250 μl    NP40 detergent by pipetting up and down several times. Transfer to a    pre-chilled microcentrifuge tube. Incubate for 15 minutes on ice.-   2. Vortex 10 seconds at highest setting.-   3. Centrifuge suspension for 30 seconds at 2-3,000×g in a centrifuge    pre-cooled at 4° C.-   4. Transfer supernatant (cytoplasmic fraction) into a pre-chilled    microcentrifuge tube. (If you began working from tissue, combine    this supernatant with that obtained in Step 1, No. 3 of the Nuclear    enrichment protocol for tissue.) Store the supernatant at −80° C.    until ready to use. Use the pellet for nuclear collection.

Step 3: Nuclear enrichment Collection and nuclear extracts.

-   1. Resuspend nuclear pellet in fixation and permeabilizing buffer or    500 μl Total lysis buffer by pipetting up and down. Vortex 10    seconds at highest setting.-   2. Incubate suspension for 30 minutes on ice on a rocking platform    set at 150 rpm.-   3. Vortex 30 seconds at highest setting. Centrifuge for 10 minutes    at 14,000×g in a microcentrifuge pre-cooled at 4° C. Transfer    supernatant (nuclear fraction) into a pre-chilled microcentrifuge    tube.-   4. Aliquot and store at −80° C. Avoid freeze/thaw cycles.

Starting from Tissue:

Step 1: Tissue Homogenization

-   1. Weigh tissue and dice into very small pieces using a clean razor    blade. Collect pieces in a pre-chilled, clean MP homogenizer    Fastprep 24 sample prep tubes (6900 series).-   2. On ice, add 3 ml ice-cold 1× hypotonic buffer supplemented with    DTT and Detergent    (3 μl of the provided 1 M DTT and 3 μl of the provided detergent)    per gram of tissue and homogenize. Incubate on ice for 15 minutes.-   3. Centrifuge for 10 minutes at 850×g at 4° C. Transfer the    supernatant into a pre-chilled microcentrifuge tube. (Save this    supernatant and pool it with the supernatant that will be collected    later in Step 2, No. 3 of the Nuclear enrichment protocol for    cells.)-   4. At this point, the tissue is homogenized. However, most of the    cells are not yet lysed. Therefore, continue the procedure with the    cell pellet at Step 2, No. 1 of the Nuclear enrichment protocol for    cells, based on a 20×10⁶ cells.

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What is claimed is:
 1. A method of multiplex detecting a first protein-second protein interaction, in a sample, for up to at least four distinct first proteins, first proteins A, B, C and D respectively, the method comprising: contacting the sample with a (i) a first agent attached to a surface of a magnetic bead that is not labeled with a first primary optically-active label, and (ii) a second primary agent attached to a surface of a magnetic bead that is labeled with a first primary optically-active label, and (iii) a third primary agent attached to a surface of a non-magnetic bead that is not labeled with a second primary optically-active label, and (iv) a fourth primary agent attached to the surface of a non-magnetic bead that is labeled with a second primary optically-active label, wherein the first, second, third and fourth primary agents are different agents each capable of capturing the distinct first proteins A, B, C and D, respectively; contacting captured first protein-second protein complex(es) with a plurality of secondary agents, each of the plurality being specific for a distinct second protein, and each labeled with a separate secondary optically-active label wherein the secondary optically-active labels are not the same as the primary optically-active labels of the primary agents and are each distinct from the secondary optically-active label of every other of the optically-active labeled secondary agents; recovering magnetic beads complexes from the sample by applying a magnetic field; recovering non-magnetic bead complexes from the sample based on a non-magnetic physical property of the non-magnetic beads; passing the recovered magnetic bead complexes through a flow cytometer or optical plate reader; passing the recovered non-magnetic bead complexes through a flow cytometer or optical plate reader; detecting the optical signal(s) of the recovered magnetic bead complexes; and detecting the optical signal(s) of the recovered non-magnetic bead complexes; wherein the presence on a magnetic bead complex of only a secondary optically-active label indicates the interaction between the first protein A and a second protein corresponding to the secondary optically-active labeled secondary agent, and wherein the presence on a magnetic bead complex of both (i) a first primary optically-active label and (ii) a secondary optically-active label indicates the interaction of the first protein B and a second protein corresponding to the secondary optically-active labeled secondary agent, and wherein the presence on a non-magnetic bead complex of only a secondary optically-active label indicates the interaction of the first protein C and a second protein corresponding to the secondary optically-active secondary labeled agent, and wherein the presence on a non-magnetic bead complex of both (i) a second primary optically-active label and (ii) a secondary optically-active label indicates the interaction of the first protein D and a second protein corresponding to the secondary optically-active labeled secondary agent.
 2. A method of multiplex detecting protein-nucleic acid interactions in a sample for up to at least four distinct proteins, proteins A, B, C and D respectively, the method comprising: a) contacting the sample with a (i) a first agent attached to a surface of a magnetic bead that is not labeled with a first primary optically-active label, and (ii) a second primary agent attached to a surface of a magnetic bead that is labeled with a first primary optically-active label, and (iii) a third primary agent attached to a surface of a non-magnetic bead that is not labeled with a second primary optically-active label, and (iv) a fourth primary agent attached to the surface of a non-magnetic bead that is labeled with a second primary optically-active label, wherein the first, second, third and fourth primary agents are different agents each capable of capturing the distinct proteins A, B, C and D, respectively, under conditions which permit capturing to the primary agents a first protein-nucleic acid complex from the sample; b) recovering magnetic beads complexes from the sample by applying a magnetic field and recovering non-magnetic bead complexes from the sample based on a non-magnetic physical property of the non-magnetic beads; c) contacting one or more of (i) the magnetic bead complexes not having a first primary optically-active label; (ii) the magnetic bead complexes having a first primary optically-active label; (iii) the non-magnetic bead complexes not having a first primary optically-active label; (iv) the non-magnetic bead complexes having a first primary optically-active label, with a Proteinase K so as to digest the proteins thereon and release any nucleic acids bound thereto; d) sequencing nucleic acid(s) released in step c)(i) so as to thereby identify the nucleic acids that have interacted with distinct protein A; in step c)(ii) so as to thereby identify the nucleic acids that have interacted with distinct protein B; in step c)(iii) so as to thereby identify the nucleic acids that have interacted with distinct protein C; and/or in step c)(iv) so as to thereby identify the nucleic acids that have interacted with distinct protein D.
 3. The method of claim 2, further comprising probing the protein-nucleic acid complex(es) with one or more optically active secondary agents each specific for one of distinct proteins A, B, C and D, so as to identify bead complexes comprising a bead, a distinct protein and a primary agent, and recovering such bead complexes.
 4. The method of claim 3, further comprising after step c) and before step d) passing the recovered magnetic bead complexes through a flow cytometer or optical plate reader and passing the recovered non-magnetic bead complexes through a flow cytometer or optical plate reader; and detecting the optical signal(s) of the recovered magnetic bead complexes and detecting the optical signal(s) of the recovered non-magnetic bead complexes and, optionally, quantifying the optical signal(s) detected so as to thereby quantify the amount of protein-nucleic acid interaction on the bead.
 5. The method of claim 2, 3 or 4, further comprising amplifying the nucleic acids released after contacting with a Proteinase K, but prior to sequencing.
 6. The method of any of claims 1-5, wherein the presence on a bead complex of a first primary optically-active label and/or a secondary optically-active label is determined by quantifying the optical signal thereof.
 7. The method of claim 5, wherein the optical signal is collected with one or more photomultipliers.
 8. The method of any of claims 1, 6 or 7, further comprising quantifying the optical signal(s) detected so as to thereby quantify the amount of first protein-second protein interaction on the bead and, optionally, comparing the quantified amount against a control amount or control curve.
 9. The method of any of claims 1-7, wherein each primary agent comprises an antibody or comprises an antigen-binding fragment of an antibody.
 10. The method of any of claims 1 or 6-9, wherein each secondary agent comprises an antibody or comprises an antigen-binding fragment of an antibody.
 11. The method of any of claims 1-10, wherein the sample is a cell or tissue lysate.
 12. The method of any of claims 1-11, wherein the sample is a cell, and the cell is fixed and permeabilized so as to permit primary and second agent entry into the cell prior to performing the method.
 13. The method of any of claims 1 or 6-12, wherein the first protein-second protein complex comprises a protein of that is a product of a gene variant.
 14. The method of any of claims 1 or 6-12, wherein the first proteins A, B, C and D are distinct variant forms of a single protein.
 15. The method of any of claims 2-5, wherein the proteins A, B, C and D are distinct variant forms of a single protein.
 16. The method of any of claims 1-15, wherein FSC and/or SSC are adjusted with a control un-complexed bead population prior to initiating the method so as to permit complexed beads to be detected.
 17. The method of any of claims 1-16, wherein the ratio of primary agent to primary optically-active labels on primary agents so-labeled is 1:1.
 18. The method of any of claims 1 or 6-17, wherein the ratio of secondary agent to secondary optically-active labels on secondary agents so-labeled is 1:1.
 19. The method of any of claims 9-10, wherein the antibodies are monoclonal antibodies.
 20. The method of any of claims 9-10 or 19, wherein the antibody fragments are F(ab′)₂ fragments, Fab′ fragments or ScFvs.
 21. The method of any of claims 1-20, wherein the magnetic beads are epoxy-coated magnetic beads.
 22. The method of any of claims 1-21, wherein the non-magnetic beads are carboxyl modified beads.
 23. The method of any of claims 1-22, wherein the agents are attached to the beads by covalent binding.
 24. The method of any of claims 1-23, wherein optically-active labels are chosen from the group of fluorophores and nanocrystals.
 25. The method of any of claims 1-24, wherein the sample is a lysate and magnetic beads are separated from the lysate using a magnetic field.
 26. The method of any of claims 1-25, wherein the sample is a lysate and the non-magnetic beads are separated from the lysate by centrifugation.
 27. The method of any of claims 1-26, wherein quantitatively measured optical activity of labeled agents bound to the complex is converted into a relative or an absolute quantitation number of co-binding molecules in each complex.
 28. The method of any of claims 1-27, wherein forward scatter amplitude gain and side scatter voltage on a flow cytometer are set to register populations of bead events to on scale, followed by applying an inclusion gate where selected linear populations of beads form collective clusters containing interrogation targets can be analyzed in their entirety by flow cytometry.
 29. The method of any of claims 1-28, wherein the cell or tissue lysate is from primary isolated cells, lymphoblasts, fibroblasts, cancer cells, a cell line, transfected cells, tissue or blood.
 30. A kit for detecting changes in protein expression in cells and for analysis of gene variants, the kit comprising: magnetic beads for immunoprecipitation, non-magnetic beads for immunoprecipitation, a lysis formulation, one or more Proteinase K inhibitors, one or more phosphatase inhibitors, a coupling buffer, nucleic acids recovery elution buffer, one or more functional variant assay (FVA) buffers, a Western loading buffer, one or more optically active labels, and instructions for use of the kit.
 31. The kit of claim 30, further comprising one or more of: a nucleic acid recovery buffer, a proteinase inhibitor, a fix-permeabilization buffer, and one or more primary agents for capturing protein-protein complexes or protein-nucleic acid complexes.
 32. The kit of claim 30 or 31, wherein the optically active labels are nanoparticles and/or fluorescent dyes.
 33. The kit of any of claims 30-32, wherein primary agents are attached to the beads.
 34. The kit of any of claims 30-33, wherein a portion and/or subset of the beads are labeled with an optically active agent.
 35. A method of detecting and analyzing a gene variant based on a protein-protein interaction, the method comprising: attaching a first primary agent to the surface of magnetic beads that are not labeled with an optically active label and attaching a second primary agent to the surface of magnetic beads that are labeled with an optically active label; attaching a third primary agent to the surface of non-magnetic beads that are not labeled with an optically active label and attaching a fourth primary agent to the surface of non-magnetic beads that are labeled with an optically active label, wherein the first, second, third and fourth primary agents are different agents and wherein the first, second, third and fourth primary agents are each capable of capturing a distinct protein complex from a cell or tissue lysate; capturing to the primary agents a protein complex from a cell or tissue lysate, where the protein complex comprises a protein of interest that is a product of a gene or a gene variant and where the protein of interest forms part of a complex with another protein; probing the protein-protein complex with one or more optically active secondary agents specific for a member of the complex; wherein the same one or more optically active labels can be used to label secondary agents on any of i) the magnetic beads that are not labeled with an optically active label, ii) the magnetic beads that are labeled with an optically active label, iii) the non-magnetic beads that are not labeled with an optically active label, and iv) the non-magnetic beads that are labeled with an optically active label; separating protein-magnetic bead complexes from the lysate based on magnetic properties of the magnetic beads; separating protein-non-magnetic bead complexes from the lysate based on a physical property of the non-magnetic beads; and measuring optical activity of optically active-labeled agents on the protein-bead complexes, wherein the absence or presence of the optically active label on the magnetic beads is used to distinguish optically active protein complexes captured by the first and second primary agents, respectively, and wherein the absence or presence of the optically active label on the non-magnetic beads is used to distinguish optically active protein complexes captured by the third and fourth primary agents, respectively.
 36. A method of detecting and/or analyzing a gene variant based on a protein-nucleic acid interaction, the method comprising: attaching a first primary agent to the surface of magnetic beads that are not labeled with an optically active label and attaching a second primary agent to the surface of magnetic beads that are labeled with an optically active label; attaching a third primary agent to the surface of non-magnetic beads that are not labeled with an optically active label and attaching a fourth primary agent to the surface of non-magnetic beads that are labeled with an optically active label, wherein the first, second, third and fourth primary agents are different agents and wherein the first, second, third and fourth primary agents are each capable of capturing a distinct protein-nucleic acid complex from a cell or tissue lysate; capturing to the primary agents a protein-nucleic acid complex from a cell or tissue lysate, where the protein-nucleic acid complex comprises a gene or a gene variant nucleic acid sequence; separating protein-nucleic acid-magnetic bead complexes from the lysate based on magnetic properties of the magnetic beads; separating protein-nucleic acid-non-magnetic beads complexes from the lysate based on a physical property of the non-magnetic beads; digesting proteins on the protein-nucleic acid bead complexes to release nucleic acids; and amplifying the released nucleic acids; wherein the absence or presence of the optically active label on the magnetic beads is used to distinguish optically active protein-nucleic complexes captured by the first and second primary agents, respectively, and wherein the absence or presence of the optically active label on the non-magnetic beads is used to distinguish optically active protein-nucleic acid complexes captured by the third and fourth primary agents, respectively.
 37. A kit for obtaining nuclear, cytoplasmic or whole-cell extract from cells or from tissue, the kit comprising: cell lysis buffer 1 M Dithiothreitol (DTT) one or more phosphatase inhibitors 10×PBS one or more phosphatase inhibitors 10× hypotonic buffer detergent written instructions for use of the kit. 